Thermo NanoDrop One User Manual

NanoDrop Micro-UV/Vis Spectrophotometers

NanoDrop One

User Guide

269-309101 Revision B July 2016

DYMO and LabelWriter are either trademarks or registered trademarks of Newell Rubbermaid in the United States and/or other countries. Wi-Fi is either a trademark or a registered trademark of Wi-Fi Alliance in the United States and/or other countries. Bluetooth is either a trademark or a registered trademark of Bluetooth Special Interest Group. Windows is either a trademark or a registered trademark of Microsoft Corporation in the United States and/or other countries. All other trademarks are the property of Thermo Fisher Scientific inc. and its subsidiaries.

For U.S. Technical Support, please contact:

Unity Lab Services Part of Thermo Fisher Scientific 5225 Verona Road

For International Support, please contact:

Thermo Fisher Scientific Telephone: +1 608 273 5017 E-mail: support.madison@thermofisher.com

Madison WI 53711-4495 U.S.A. Telephone: 1 800 532 4752 E-mail: us.techsupport.analyze@thermofisher.com

Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.

The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser.

This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents.

For Research Use Only. This instrument or accessory is not a medical device and is not intended to be used for the prevention, diagnosis, treatment or cure of disease.

WARNING Avoid an explosion or fire hazard. This instrument or accessory is not designed for use in an explosive atmosphere.

Chapter 1 About the NanoDrop One Spectrophotometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Instrument Models and Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Optional Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Update Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Detection Limits for All Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Measure dsDNA, ssDNA or RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Measure dsDNA, ssDNA or RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Nucleic Acid Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Setting for Nucleic Acid Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Calculations for Nucleic Acid Measurements. . . . . . . . . . . . . . . . . . . . . . . . . 18

Measure Microarray. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Measure Microarray Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Microarray Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Settings for Microarray Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Calculations for Microarray Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Measure using a Custom Factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Measure Nucleic Acid using a Custom Factor . . . . . . . . . . . . . . . . . . . . . . . . 35

Custom Factor Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Settings for Nucleic Acid Measurements using a Custom Factor . . . . . . . . . . 39

Detection Limits for Nucleic Acid Measurements using a Custom

Factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Measure Oligo DNA or Oligo RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Measure Oligo DNA or Oligo RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Oligo Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Settings for Oligo DNA and Oligo RNA Measurements . . . . . . . . . . . . . . . . 47

Detection Limits for Oligo DNA and Oligo RNA Measurements . . . . . . . . . 48

Calculations for Oligo DNA and Oligo RNA Measurements . . . . . . . . . . . . 49

Thermo Scientific NanoDrop One User Guide iii

Contents

Measure Protein A280. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Measure Protein Concentration at A280 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Protein A280 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Settings for Protein A280 Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Detection Limits for Protein A280 Measurements. . . . . . . . . . . . . . . . . . . . . 64

Calculations for Protein A280 Measurements . . . . . . . . . . . . . . . . . . . . . . . . 65

Measure Proteins and Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Measure Labeled Protein Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Proteins & Labels Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Settings for Proteins and Labels Measurements . . . . . . . . . . . . . . . . . . . . . . . 74

Detection Limits for Proteins and Labels Measurements . . . . . . . . . . . . . . . . 76

Calculations for Proteins and Labels Measurements. . . . . . . . . . . . . . . . . . . . 77

Measure Protein A205. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Measure Protein Concentration at A205 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Protein A205 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Settings for Protein A205 Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Calculations for Protein A205 Measurements . . . . . . . . . . . . . . . . . . . . . . . . 85

Measure Protein BCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Protein BCA Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Settings for Protein BCA Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Measure Protein Bradford . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Protein Bradford Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Settings for Protein Bradford Measurements . . . . . . . . . . . . . . . . . . . . . . . . 109

Measure Protein Lowry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Protein Lowry Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Settings for Protein Lowry Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Measure Protein Pierce 660. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Protein Pierce 660 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Settings for Protein Pierce 660 Measurements . . . . . . . . . . . . . . . . . . . . . . . 127

Measure OD600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Measure OD600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

OD600 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Settings for OD600 Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Calculations for OD600 Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Measure Custom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Measure using a Custom Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Delete Custom Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Custom Method Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

iv NanoDrop One User Guide Thermo Scientific

Contents

Measure UV-Vis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Measure UV-Vis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

UV-Vis Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Settings for UV-Vis Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Measure Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Measure Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Create Kinetics Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Edit Kinetics Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Kinetics Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Settings for Kinetic Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Chapter 3 Learning Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175

Micro-Volume Sampling—How it Works. . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Set Up the Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Measure a Micro-Volume Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Measure a Sample Using a Cuvette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Prepare Samples and Blanks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

Basic Instrument Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

NanoDrop One Home Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

NanoDrop One Measurement Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

NanoDrop One Data Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

NanoDrop One General Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

Acclaro Sample Intelligence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

NanoDrop One Viewer Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Viewer Home Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Manage Experiments and Associated Data. . . . . . . . . . . . . . . . . . . . . . . . . . 251

Manage Identifiers on a PC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

Manage Custom Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

Multimedia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Chapter 4 Maintaining Your Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .279

Maintenance Schedule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

Cleaning the Touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Maintaining the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

Cleaning the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

Reconditioning the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

Decontaminating the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

Maintaining the Cuvette Sampling System . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

Instrument Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Intensity Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Performance Verification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Pedestal Image Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

Thermo Scientific NanoDrop One User Guide v

Contents

Chapter 5 Safety and Operating Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .301

Operating Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

Safety Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Chapter 6 About this Help System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .311

Chapter 7 Contact Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .313

vi NanoDrop One User Guide Thermo Scientific

About the NanoDrop One Spectrophotometer

The Thermo Scientific™ NanoDrop™ One is a compact, stand-alone UV-Visible spectrophotometer developed for micro-volume analysis of purified nucleic acids and a wide variety of proteins. The patented sample retention system enables the measurement of highly concentrated samples without the need for dilutions.

The NanoDrop One system comes with preloaded software and a touchscreen display. The instrument can be connected to an optional USB label printer.

NOTICE Before operating a NanoDrop One instrument, please read the safety and

operating precautions and then follow their recommendations when using the instrument.

Instrument Models and Features

There are two models available for the NanoDrop One spectrophotometer...

Optional Accessories

A number of accessories are available for the NanoDrop One instruments...

Thermo Scientific NanoDrop One User Guide 1

Update Software

Quickly and easily download the latest NanoDrop One software...

NanoDrop One Spectrophotometer NanoDrop OneC Spectrophotometer

Arm

Pedestal

Cuvette holder

About the NanoDrop One Spectrophotometer

Instrument Models and Features

There are two models available for the NanoDrop One spectrophotometer—the NanoDrop One and the NanoDrop One models include the patented

micro-volume sample retention system and general

features. The

NanoDrop One

features a cuvette holder for analyzing dilute samples using standard UV-visible cuvettes.

Both instruments come with a built-in, 7-inch Android high-resolution touchscreen preloaded with easy-to-use instrument control software. The NanoDrop One software is loaded with features to integrate with and simplify your daily workflows.

. Both

model also

Locate the instrument away from air vents and exhaust fans to minimize evaporation

2 NanoDrop One User Guide Thermo Scientific

Touchscreen

USB-A port

Pedestals

Touchscreen

About the NanoDrop One Spectrophotometer

Instrument Models and Features

Touchscreen can slide left or right to accommodate personal preference, and tilt forward or back for optimal viewing

Two more USB-A ports are located on instrument back panel

Thermo Scientific NanoDrop One User Guide 3

Instrument light path

Cuvette holder

About the NanoDrop One Spectrophotometer

Instrument Models and Features

NanoDrop One Software with Acclaro Sample Intelligence Technology

The Thermo Scientific™ Acclaro™ Sample

Intelligence technology built into the

NanoDrop One instruments provides these exclusive features to help you assess sample integrity:

• contaminant analysis to help qualify a sample before use in downstream applications

• on-demand technical support for measurements that are atypical or very low concentration

• invalid result alerts (a column sensor monitors for the presence of bubbles or reflective particles that can compromise measurement results)

NanoDrop OneC Model Additional Features

The NanoDrop One includes a cuvette holder for measuring dilute samples, colorimetric assays, cell cultures and kinetic studies. The cuvette system has these additional features:

• extended lower detection

limits

• 37 °C heater option for temperature-sensitive samples and analyses

• micro-stirring option to ensure sample homogeneity and support kinetic studies

For details, see Measure a

Sample using a Cuvette.

model

4 NanoDrop One User Guide Thermo Scientific

Optional Accessories

A number of accessories are available for the NanoDrop One instruments. To order an accessory, contact your local distributor or visit our website.

DYMO™ LabelWriter™ 450 USB Label Printer

Prints two 5/16-in x 4-in self-adhesive labels for transferring sample data directly into laboratory notebooks or posting on bulletin boards. The software allows printing of data from each sample measurement or from a group of samples logged and measured together.

The printer connects to the instrument (front or back) via a USB cable (included).

PR-1 Pedestal Reconditioning Kit

About the NanoDrop One Spectrophotometer

Optional Accessories

Specially formulated conditioning compound that can be applied to the pedestals to restore them to a hydrophobic state (required to achieve adequate surface tension for accurate sample measurements). The kit includes conditioning compound and applicators. For more information, see

Reconditioning the Pedestals.

PV-1 Performance Verification Solution

Liquid photometric standard used to check instrument performance. For more information, see Performance Verification.

Thermo Scientific NanoDrop One User Guide 5

About the NanoDrop One Spectrophotometer

Register Your Instrument

Register your instrument to receive e-mail updates on software and accessories for the NanoDrop One instruments. An Internet connection is required for registration.

 To register your instrument

1. Do one of the following:

–From the NanoDrop One Viewer software running on a personal

computer (PC) that is connected to the Internet, open the Help menu and choose NanoDrop One Website.

– From any PC that is connected to the Internet, use any web browser to

navigate to our website.

2. On the website, locate NanoDrop One Registration and follow the instructions to register the instrument.

6 NanoDrop One User Guide Thermo Scientific

Update Software

About the NanoDrop One Spectrophotometer

Quickly and easily download and install the latest NanoDrop One software and release notes from our website. Follow the steps to update or upgrade the software on your local instrument and/or install or update the NanoDrop One Viewer software on a personal computer (PC). An Internet connection is required to download software.

Update Software

To install or update NanoDrop One Viewer software

1. Do one of the following:

• To install the Viewer software on a computer for the first time, open any

web browser and find the NanoDrop website.

• To update or upgrade the Viewer software, from the Viewer Home screen,

open the Help menu and choose NanoDrop One Website to open our website.

2. On the NanoDrop website, locate the software downloads page.

3. Select to download NanoDrop One (PC) Viewer software (English version) and follow the instructions to download and run the installer. (A computer restart is required after the installer completes.)

4. To add a language, including software and Help systems, download and run the language pack installer (English must be installed first). (No computer restart is required after a language installer completes.)

Thermo Scientific NanoDrop One User Guide 7

About the NanoDrop One Spectrophotometer

Update Software

To update or upgrade NanoDrop One instrument software

1. Do one of the following:

2. Insert a USB device such as a memory stick into a USB port on the computer.

3. On the NanoDrop website, locate the software downloads page, select to

4. To add a language, including software and Help systems, download the

5. Insert the USB device into any USB port on the NanoDrop One instrument.

–From the NanoDrop One Viewer software, open the Help menu and

choose NanoDrop One Website to open our website.

– From any personal computer that is connected to the Internet, navigate to

the NanoDrop website.

update or upgrade NanoDrop One operating software (English version) and follow the instructions to download the installer to the USB device.

language pack installer(s) to the USB device.

6. From the instrument Home screen, tap (Settings) > System > Update Software.

If the USB device contains more than one version of the installer, a message is displayed. Select the version to install (English installer must be run first) and tap Update. (An instrument restart is required after the English installer completes.)

When the installation is complete, a message similar to the following appears next to the Update Software button:

Version: 1.2.0 (currently installed version of instrument operating software) Database version: 1 (version of NanoDrop One database on this instrument)

7. To add a language, including software and Help systems, tap Update Software again, select the language and version to install and tap Update. (No instrument restart is required after a language installer completes.)

Note: To change the language, tap Language, select an installed language and tap OK. (An instrument restart is required after you change the language.)

8 NanoDrop One User Guide Thermo Scientific

Applications

Detection Limits for All Applications

Note Detection limits provided in the tables below are approximate and apply to

micro-volume measurements only; they are based on the instrument’s photometric absorbance range (10 mm equivalent) of 0–550 A. For measurements with 10 mm pathlength cuvettes, the photometric absorbance range is 0–1.5 A.

Detection limits for standard applications

Sample Type Lower Detection Limit Upper Detection Limit Typical Reproducibility

dsDNA 2.0 ng/μL (pedestal)

0.20 ng/μL (cuvette)

ssDNA 1.3 ng/μL (pedestal)

0.13 ng/μL (cuvette)

RNA 1.6 ng/μL (pedestal)

0.16 ng/μL (cuvette)

Thermo Scientific NanoDrop One User Guide 9

27,500 ng/μL (pedestal)

75 ng/μL (cuvette)

18,150 ng/μL (pedestal)

49.5 ng/μL (cuvette)

22,000 ng/μL (pedestal)

60 ng/μL (cuvette)

±2.0 ng/μL for sample concentrations between 2.0 and 100 ng/μL samples; ±2% for samples >100 ng/μL

Applications

Detection Limits for All Applications

Sample Type Lower Detection Limit Upper Detection Limit Typical Reproducibility

DNA Microarray (ssDNA)

Purified BSA by Protein A280

IgG by Protein A280

Purified BSA by

1.3 ng/μL (pedestal)

0.13 ng/μL (cuvette)

0.06 mg/mL (pedestal)

0.006 mg/mL (cuvette)

0.03 mg/mL (pedestal)

0.003 mg/mL (cuvette)

0.06 mg/mL (pedestal)

495 ng/μL (pedestal)

±2.0 ng/μL for sample concentrations between 2.0 and 100 ng/μL samples;

49.5 ng/μL (cuvette)

825 mg/mL (pedestal)

±2% for samples >100 ng/μL

±0.10 mg/mL (for 0.10–10 mg/mL samples); ±2% for samples >10 mg/mL

402 mg/mL (pedestal)

19 mg/mL (pedestal) ±0.10 mg/mL for 0.10–10 mg/mL samples

Proteins & Labels

0.006 mg/mL (cuvette)

Protein BCA 0.2 mg/mL (20:1

8.0 mg/mL (pedestal)

2% over entire range

reagent/sample volume)

0.20 mg/mL (cuvette)

0.01 mg/mL over entire range

0.01 mg/mL (1:1 reagent/sample volume)

Protein Lowry 0.2 mg/mL (pedestal) 4.0 mg/mL (pedestal) 2% over entire range

Protein Bradford 100 μg/mL (50:1

reagent/sample volume)

8000 μg/mL

±25 μg/mL for 100–500 μg/mL samples ±5% for 500–8000 μg/mL samples

15 μg/mL (1:1 reagent/sample volume)

Protein Pierce 660 50 μg/mL (15:1

reagent/sample volume)

25 μg/mL (7.5:1 reagent/sample volume)

Based on five replicates (SD=ng/μL; CV=%)

Note To minimize instrument error with highly concentrated samples, make dilutions to ensure that measurements are made within these absorbance limits:

• For micro-volume measurements, maximum absorbance at 260 nm (for nucleic acids) or 280 nm (for proteins) should be less than 62.5 A.

• For measurements with 10 mm pathlength cuvettes, maximum absorbance at 260 nm (or 280 nm for proteins) should be less than 1.5 A, which is approximately 75 ng/μL dsDNA.

100 μg/μL

2000 μg/mL

1000 μg/mL

±4 μg/mL for 15–50 μg/mL samples ±5% for 50–125 μg/mL samples

±3 μg/mL for 50–125 μg/mL samples ±2% for samples > 125 μg/mL

±3 μg/mL for 25–125 μg/mL samples ±2% for samples >125 μg/mL

10 NanoDrop One User Guide Thermo Scientific

Detection limits for pre-defined dyes

Applications

Detection Limits for All Applications

Sample Type Lower Detection Limit Upper Detection Limit

Cy3, Cy3.5, Alexa Fluor

0.2 pmol/μL (pedestal) 100 pmol/μL (pedestal) ±0.20 pmol/μL for sample

555, Alexa Fluor 660

Typical Reproducibility

concentrations between 0.20 and 4.0

pmol/μL; ±2% for samples >4.0 pmol/μL

Cy5, Cy5.5, Alexa Fluor 647

0.12 pmol/μL (pedestal) 60 pmol/μL (pedestal) ±0.12 pmol/μL for sample concentrations

between 0.12 and 2.4 pmol/μL; ±2% for samples >2.4 pmol/μL

Alexa Fluor 488, Alexa Fluor 594

0.4 pmol/μL (pedestal) 215 pmol/μL (pedestal) ±0.40 pmol/μL for sample concentrations

between 0.40 and 8.0 pmol/μL; ±2% for samples >8.0 pmol/μL

Alexa Fluor 546 0.3 pmol/μL (pedestal) 145 pmol/μL (pedestal) ±0.30 pmol/μL for sample concentrations

between 0.30 and 6.0 pmol/μL; ±2% for samples >6.0 pmol/μL

Values are approximate

Based on five replicates (SD=ng/μL; CV=%)

Thermo Scientific NanoDrop One User Guide 11

Measure dsDNA, ssDNA or RNA

Measures the concentration of purified dsDNA, ssDNA or RNA samples that absorb at 260 nm.

Measure dsDNA, ssDNA or RNA

Reported Results

Settings

Detection Limits

Calculations

Measure dsDNA, ssDNA or RNA

Use the dsDNA, ssDNA and RNA applications to quantify purified double-stranded (ds) or single-stranded (ss) DNA or RNA samples. These applications report nucleic acid concentration and two absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction can also be used.

To measure dsDNA, ssDNA or RNA samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop One User Guide 13

Measure dsDNA, ssDNA or RNA

Typical nucleic acid spectrum

Comparison of nucleic acid spectra with and without two common contaminants

Before you begin...

Before taking pedestal measurements with the NanoDrop One instrument, lift the instrument arm and clean the upper and lower pedestals. At a minimum, wipe the pedestals with a new laboratory wipe. For more information, see Cleaning the Pedestals.

 To measure nucleic acid

1. From the Home screen, select the Nucleic Acids tab and tap dsDNA, ssDNA or RNA, depending on the samples to be measured.

2. Specify a baseline correction if desired.

3. Pipette 1–2 μL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette

light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 1-2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if

Auto-Measure is off, lower arm and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

14 NanoDrop One User Guide Thermo Scientific

Measure dsDNA, ssDNA or RNA

Best practices for nucleic acid measurements

• Isolate and purify nucleic acid samples before measurement to remove impurities. Depending on the sample, impurities could include DNA, RNA, free nucleotides, proteins, some buffer components and dyes. See Preparing Samples for more information.

Note Extraction reagents such as guanidine, phenol, and EDTA contribute absorbance between 230 nm and 280 nm and will affect measurement results if present in samples (even residual amounts).

• Ensure the sample absorbance is within the instrument’s absorbance detection limits.

• Blank with the same buffer solution used to resuspend the analyte of interest. The blanking solution should be a similar pH and ionic strength as the analyte solution.

•Run a blanking cycle to assess the absorbance contribution of your buffer solution. If the buffer exhibits strong absorbance at or near the analysis wavelength (typically 260 nm), you may need to choose a different buffer or application. See Choosing and Measuring a

Blank for more information.

• For micro-volume measurements:

– Ensure pedestal surfaces are properly cleaned and conditioned.

– If possible, heat highly concentrated or large molecule samples, such as genomic or

lambda DNA, to 63 °C (145 °F) and gently (but thoroughly) vortex before taking a measurement. Avoid introducing bubbles when mixing and pipetting.

– Follow best practices for micro-volume measurements.

– Use a 1-2 μL sample volume. See Recommended Sample Volumes for more

information.

• For cuvette measurements (NanoDrop One

instruments only), use compatible cuvettes

and follow best practices for cuvette measurements.

Related Topics

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

Thermo Scientific NanoDrop One User Guide 15

Measure dsDNA, ssDNA or RNA

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see

more/less sample data

Nucleic acid concentration

UV spectrum

Tap to select unit

Purity ratios

Menu of options; tap to open

Sample name;

tap to edit

Swipe screen left to

view table with more measurement results

Pinch and zoom to adjust axes; double-tap to reset

Tap to end experiment and export data

Menu of options;

tap to open

Nucleic Acid Reported Results

dsDNA measurement screen

For each measured sample, the dsDNA, ssDNA and RNA applications show the UV absorbance spectrum and a summary of the results. Here is an example:

Note Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

16 NanoDrop One User Guide Thermo Scientific

Measure dsDNA, ssDNA or RNA

dsDNA, ssDNA and RNA reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

• sample details (application and sampling method used, i.e., pedestal or cuvette)

•sample name

• created on (date sample measurement was taken)

• nucleic acid concentration

• A260/A280

• A260/A230

•A260

•A280

•factor

• baseline correction

Related Topics

• Basic Instrument Operations

• Nucleic Acid Calculations

Setting for Nucleic Acid Measurements

To show the dsDNA, ssDNA or RNA settings, from the dsDNA, ssDNA or RNA measurement screen, tap > Nucleic Acid Setup.

Thermo Scientific NanoDrop One User Guide 17

Measure dsDNA, ssDNA or RNA

Setting Available Options Description

Baseline Correction On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Optional user-defined baseline correction. Can be used to correct for any offset caused by light scattering particulates by subtracting measured absorbance at specified baseline correction wavelength from absorbance values at all wavelengths in sample spectrum. As a result, absorbance of sample spectrum is zero at specified baseline correction wavelength.

Related Topics

• Instrument Settings

Calculations for Nucleic Acid Measurements

The nucleic acid applications use the Beer-Lambert

equation to correlate absorbance with concentration.

Solving Beer’s law for concentration yields the equation at the right.

Beer-Lambert Equation (solved for concentration)

c = A / ( * b)

where:

A = UV absorbance in absorbance units (AU)

 = wavelength-dependent molar absorptivity coefficient (or extinction

coefficient) in liter/mol-cm

b = pathlength in cm

c = analyte concentration in moles/liter or molarity (M)

Note: Dividing the measured absorbance of a sample solution by its molar extinction coefficient yields the molar concentration of the sample. See

Published Extinction Coefficients for more information regarding molar

vs. mass concentration values.

18 NanoDrop One User Guide Thermo Scientific

Measure dsDNA, ssDNA or RNA

The Nucleic Acid applications use a modification of the Beer-Lambert equation (shown at right) to calculate sample concentration where the extinction coefficient and pathlength are combined and referred to as a “factor.”

For the dsDNA, ssDNA and RNA applications, the generally accepted factors for nucleic acids are used in conjunction with Beer’s Law to calculate sample concentration. For the Custom Factor application, the user-specified factor is used.

Extinction Coefficients vs Factors

Using the terms in the Beer-Lambert equation, factor (f) is defined as:

factor (f) = 1/( * b)

where:

 = wavelength-dependent molar extinction coefficient in ng-cm/μL

b = sample pathlength in cm

As a result, analyte concentration (c) is calculated as:

c = A * [1/( * b)]

c = A * f

where:

c = analyte concentration in ng/μL A = absorbance in absorbance units (A)

f = factor in ng-cm/μL (see below)

Factors Used

• dsDNA (factor = 50 ng-cm/μL)

• ssDNA (factor = 33 ng-cm/μL)

• RNA (factor = 40 ng-cm/μL)

• Custom Factor (user entered factor between 15 ng-cm/μL and 150 ng-cm/μL

Thermo Scientific NanoDrop One User Guide 19

Measure dsDNA, ssDNA or RNA

Calculated nucleic acid concentrations are based on the absorbance value at 260 nm, the factor used and the sample pathlength. A single-point baseline correction (or analysis correction) may also be applied.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Absorbance values at 260 nm, 280 nm and sometimes 230 nm are used to calculate purity ratios for the measured nucleic acid samples. Purity ratios are sensitive to the presence of contaminants in the sample, such as residual solvents and reagents typically used during sample purification.

Measured Values

Note: For micro-volume absorbance measurements and measurements

taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

A260 absorbance

• Nucleic acid absorbance values are measured at 260 nm using the normalized spectrum. This is the reported A260 value if Baseline Correction is not selected.

•If Baseline Correction is selected, the absorbance value at the correction wavelength is subtracted from the absorbance at 260 nm. The corrected absorbance at 260 nm is reported and used to calculate nucleic acid concentration.

A230 and A280 absorbance

• Normalized and baseline-corrected (if selected) absorbance values at 230 nm and 280 nm are used to calculate A260/A230 and A260/A280 ratios.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

20 NanoDrop One User Guide Thermo Scientific

Measure dsDNA, ssDNA or RNA

Reported Values

• Nucleic acid concentration. Reported in selected unit (i.e., ng/μL, μg/uL or μg/mL). Calculations are based on modified Beer’s Law equation using corrected nucleic acid absorbance value.

• A260/A280 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm. An A260/A280 purity ratio of ~1.8 is generally accepted as “pure” for DNA (~2.0 for RNA). Acidic solutions may under represent the reported value by 0.2-0.3; the opposite is true for basic solutions.

• A260/A230 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 230 nm. An A260/A230 purity ratio between

1.8 and 2.2 is generally accepted as “pure” for DNA and RNA.

Note: Although purity ratios are important indicators of sample quality, the best quality indicator quality is functionality in the downstream application of interest (e.g., real-time PCR).

Thermo Scientific NanoDrop One User Guide 21

Measure Microarray

Measures the concentration of purified nucleic acids that have been labeled with up to two fluorescent dyes for use in downstream microarray applications.

Measure Microarray Samples

Reported Results

Settings

Detection Limits

Calculations

Measure Microarray Samples

Use the Microarray application to quantify nucleic acids that have been labeled with up to two fluorescent dyes. The application reports nucleic acid concentration, an A260/A280 ratio and the concentrations and measured absorbance values of the dye(s), allowing detection of dye concentrations as low as 0.2 picomole per microliter.

To measure microarray samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop One User Guide 23

Measure Microarray

Before you begin...

24 NanoDrop One User Guide Thermo Scientific

 To measure a microarray sample

Dye absorbance peak used to calculate dye concentration

A260 absorbance peak used to calculate nucleic acid concentration

Typical microarray spectrum

1. From the Home screen, select the Nucleic Acids tab and tap Microarray.

2. Specify the sample type and factor and the type of

dye(s) used.

Tip: Select a dye from the pre-defined list or add a custom dye using the Dye/Chromophore Editor.

3. Pipette 1–2 μL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette

light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Measure Microarray

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 1-2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if

Auto-Measure is off, lower arm and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Thermo Scientific NanoDrop One User Guide 25

Measure Microarray

Related Topics

• Best Practices for Nucleic Acid Measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

26 NanoDrop One User Guide Thermo Scientific

Microarray Reported Results

Drag tab down/up to

see more/less sample data

Dye concentration(s)

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Nucleic acid concentration

Tap to select unit

UV-visible spectrum

Menu of options; tap to open

Sample name;

tap to edit

Swipe screen left to

view table with more measurement results

Pinch and zoom to adjust axes; double-tap to reset

Tap to end experiment and export data

Microarray measurement screen

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Measure Microarray

Note

• A baseline correction is performed at 750 nm (absorbance value at 750 nm is subtracted from absorbance values at all wavelengths in sample spectrum).

• Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

Thermo Scientific NanoDrop One User Guide 27

Measure Microarray

Microarray reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

• sample details (application used and pedestal or cuvette)

•sample name

• created on (date sample measurement was taken)

• nucleic acid concentration

•A260

• A260/A280

• dye 1/dye 2 concentration

• sample type

• analysis correction

•factor

Related Topics

• Basic Instrument Operations

• Microarray Calculations

Settings for Microarray Measurements

Microarray settings

The Microarray Setup screen appears after you select the Microarray application from the Nucleic Acids tab on the Home screen. To show the Microarry settings from the Microarray measurement screen, tap > Microarray Setup.

28 NanoDrop One User Guide Thermo Scientific

Setting Available Options Description

Measure Microarray

Sample type and Factor

Dye 1/Dye 2 Type

dsDNA (with non-editable factor of 50 ng-cm/μL)

ssDNA (with non-editable factor of 33 ng-cm/μL)

RNA (with non-editable factor of 40 ng-cm/μL)

Oligo DNA with non-editable calculated factor in ng-cm/μL

Oligo RNA with non-editable calculated factor in ng-cm/μL

Custom (with user-specified factor in ng-cm/μL)

Cy3, 5, 3.5, or 5.5, Alexa Fluor 488, 546, 555, 594, 647, or 660

Widely accepted value for double-stranded DNA

Widely accepted value for single-stranded DNA

Widely accepted value for RNA

Factor calculated from user-defined DNA base sequence. When selected, available DNA base units (i.e., G, A, T, C) appear as keys. Define sequence by tapping appropriate keys. Factor is calculated automatically based on widely accepted value for each base unit.

Factor calculated from user-defined RNA base sequence. When selected, available RNA base units (i.e., G, A, U, C) appear as keys. Define sequence by tapping appropriate keys. Factor is calculated automatically based on widely accepted value for each base unit.

Enter factor between 15 ng-cm/μL and 150 ng-cm/μL

Select pre-defined dye(s) used to label sample material, or one that has been added using Dye/Chrom. Editor.

Dye 1/Dye 2 Unit picomoles/microliter (pmol/uL),

micromoles (uM), or millimoles (mM)

Analysis Correction

On or off

Enter analysis correction wavelength in nm or use default value (340 nm)

To add a custom dye or edit the list of available dyes, use the Dye/Chromophore Editor.

The Analysis Correction affects the calculation for nucleic acid concentration only.

Select unit for reporting dye concentrations

Corrects sample absorbance measurement for any offset caused by light scattering particulates by subtracting absorbance value at specified analysis correction wavelength from absorbance value at analysis wavelength. Corrected value is used to calculate sample concentration.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Analysis Correction.

Thermo Scientific NanoDrop One User Guide 29

Measure Microarray

Tap to add custom dye

Tap to edit selected custom dye

Tap to delete selected custom dye

Locked dye (pre-defined; cannot be edited or deleted)

Selected dyes (will appear in Dye1 and Dye2 lists in Microarray Setup or Proteins & Labels Setup)

Tap to close Dye/Chrom. Editor

Custom dye (user-defined; can be edited or deleted)

Dye/Chromophore Editor

Dye/chromophore editor

Use the Dye/Chromophore Editor to add a custom dye to the list of available dyes in

Microarray Setup or Proteins & Labels Setup. You can also specify which dyes are available in

that list.

To access the Dye/Chromophore Editor:

• from the Home screen, tap > Dye/Chrom. Editor

• from the Microarray or Proteins & Labels measurement screen, tap > Settings

> Dye/Chrom. Editor

30 NanoDrop One User Guide Thermo Scientific

These operations are available from the Dye/Chromophore Editor:

Measure Microarray

Add or remove a dye

To add or remove a dye from the Dye1 or Dye2 drop-down list in Microarray Setup or

Proteins & Labels Setup:

– select or deselect corresponding checkbox

Add custom dye

– tap to show New Dye box

– enter unique Name for new dye (tap field to display keyboard, tap Done key to close

keyboard)

–select default Unit that will be used to display dye concentration

–enter dye’s Extinction Coefficient (or molar absorptivity constant) in L/mole-cm

(typically provided by dye manufacturer)

–specify Wa ve le ng th in nm (between 450 nm and 700 nm) that will be used to

measure dye’s absorbance

– specify dye’s correction values at 260 nm and 280 nm

–tap Add Dye

Note To determine dye correction values (if not available from dye manufacturer):

– use instrument to measure pure dye and note absorbance at 260 nm, 280 nm

and at analysis wavelength for dye (see above)

– calculate ratio of A

260/Adye wavelength

and enter that value for 260 nm

Correction

– calculate ratio of A

280/Adye wavelength

and enter that value for 280 nm

Correction

When a custom dye is selected before a measurement, the dye’s absorbance and concentration values are reported and the corrections are applied to the measured sample absorbance values, and to the resulting sample concentrations and purity ratios.

Edit custom dye

Tip Dyes pre-defined in the software cannot be edited.

– tap to select custom dye

–tap

Thermo Scientific NanoDrop One User Guide 31

Measure Microarray

– edit any entries or settings

–tap Save Dye

Delete custom dye

Tip Dyes pre-defined in the software cannot be deleted.

– tap to select custom dye

–tap

NOTICE Deleting a custom dye permanently removes the dye and all associated information from the software.

Related Topics

• Instrument Settings

Calculations for Microarray Measurements

As with the other nucleic acid applications, the Microarray application uses a modification of the

Beer-Lambert equation to calculate sample

concentration where the extinction coefficient and pathlength are combined and referred to as a “factor.” The Microarray application offers six options (shown at right) for selecting an appropriate factor for each measured sample, to be used in conjunction with Beer’s Law to calculate sample concentration.

If the factor is known, choose the Custom Factor option and enter the factor in ng-cm/μL. Otherwise, choose the option that best matches the sample solution.

Tip: Ideally, the factor or extinction coefficient should be determined empirically using a solution of the study nucleic acid at a known concentration using the same buffer.

Available Options for Factors

• dsDNA (factor = 50 ng-cm/μL)

• ssDNA (factor = 33 ng-cm/μL)

• RNA (factor = 40 ng-cm/μL)

• Oligo DNA (calculated from user entered DNA nucleotide sequence)

• Oligo RNA (calculated from user entered RNA nucleotide sequence)

• Custom Factor (user entered factor between 15 ng-cm/μL and 150 ng-cm/μL

Note: See Sample Type for more information.

32 NanoDrop One User Guide Thermo Scientific

Measure Microarray

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Measured Values

A260 absorbance

Note: The absorbance value at 750 nm is subtracted from all wavelengths

in the spectrum. As a result, the absorbance at 750 nm is zero in the displayed spectra. Also, for micro-volume absorbance measurements and measurements taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Nucleic acid absorbance values for all Microarray sample types are measured at 260 nm using the 750-corrected and normalized spectrum.

•If Analysis Correction is selected, the absorbance value at the correction wavelength is subtracted from the absorbance at 260 nm.

• If one or more dyes are selected, the dye correction values at 260 nm are also subtracted from the absorbance at 260 nm.

• The final corrected absorbance at 260 nm is reported and used to calculate sample concentration.

A280 absorbance

Dye concentrations are calculated from the absorbance value at the dye’s analysis wavelength, the dye’s extinction coefficient, and the sample pathlength. A sloped-line dye correction may also be used.

• 750-corrected and normalized absorbance value at 280 nm (minus the A280 dye correction) is used to calculate an A260/A280 ratio.

Dye absorbance

• Dye absorbance values are measured at specific wavelengths. See

Dye/Chromophore Editor for analysis wavelengths used.

• If Sloping Dye Correction is selected, a linear baseline is drawn between 400 nm and 750 nm and, for each dye, the absorbance value of the sloping baseline is subtracted from the absorbance value at each dye’s analysis wavelength. Baseline-corrected dye absorbance values are reported and used to calculate dye concentrations.

Dye correction

• Pre-defined dyes have known correction values for A260 and A280. See Dye/Chromophore Editor for correction values used.

• A260 dye corrections are subtracted from the A260 absorbance value used to calculate nucleic acid concentration, and from the A260 absorbance value used to calculate the A260/A280 purity ratio.

Thermo Scientific NanoDrop One User Guide 33

Measure Microarray

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

•

Reported Values

• Nucleic acid concentration. Reported in selected unit (i.e., ng/μL, μg/uL or μg/mL). Calculations are based on modified Beer’s Law equation using corrected nucleic acid absorbance value.

• Dye1/Dye2 concentration. Reported in pmol/μL. Calculations are based on Beer’s Law equation using (sloping) baseline-corrected dye absorbance value(s).

Note: Although purity ratios are important indicators of sample quality, the best indicator of DNA or RNA quality is functionality in the downstream application of interest (e.g., microarray).

Related Topics

• Calculations for Nucleic Acid Measurements

34 NanoDrop One User Guide Thermo Scientific

Measure using a Custom Factor

Measures the concentration of purified nucleic acids using a custom factor for the calculations.

Measure using Custom Factor

Reported Results

Settings

Detection Limits

Calculations

Measure Nucleic Acid using a Custom Factor

Use the Custom Factor application to quantify purified DNA or RNA samples that absorb at 260 nm with a user-defined extinction coefficient or factor. The application reports nucleic acid concentration and two absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction can also be used.

To measure nucleic acid samples using a custom factor

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop One User Guide 35

Measure using a Custom Factor

Typical nucleic acid spectrum

Before you begin...

 To measure using a custom factor

1. From the Home screen, select the Nucleic Acids tab and tap Custom Factor.

2. Enter the factor to be used for the calculations and specify a baseline correction if desired.

3. Pipette 1–2 μL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette

light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 1-2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if

Auto-Measure is off, lower arm and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

36 NanoDrop One User Guide Thermo Scientific

Related Topics

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

Custom Factor Reported Results

Measure using a Custom Factor

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Note The Custom Factor measurement screen is identical to the measurement screen for

the other nucleic acid applications except the Custom Factor is reported in the lower left

corner (see image below).

Thermo Scientific NanoDrop One User Guide 37

Measure using a Custom Factor

Custom factor used to calculate nucleic acid concentration

Related Topics

• Basic Instrument Operations

• Nucleic Acid Reported Results

• Nucleic Acid Calculations

38 NanoDrop One User Guide Thermo Scientific

Settings for Nucleic Acid Measurements using a Custom Factor

To show the Custom Factor settings, tap > Custom Factor Setup.

Setting Available Options Description

Measure using a Custom Factor

Custom Factor Enter an integer value

between 15 ng-cm/μL and 150 ng-cm/μL

Baseline Correction On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Related Topics

Constant used to calculate nucleic acid concentration in

modified Beer’s Law equation. Based on extinction coefficient

and pathlength:

f = 1/(



* b))

260

where: f= factor

 = molar extinction coefficient at 260 nm in ng-cm/μL

b = sample pathlength in cm (1 cm for nucleic acids measured with the NanoDrop One instruments)

• Instrument Settings

Detection Limits for Nucleic Acid Measurements using a Custom Factor

The lower detection limits and reproducibility specifications for nucleic acids are provided

here. The upper detection limits are dependent on the upper absorbance limit of the

instrument and the user-defined extinction coefficients.

To calculate upper detection limits for nucleic acid samples

To calculate upper detection limits in ng/μL, use the following equation:

(upper absorbance limit

Thermo Scientific NanoDrop One User Guide 39

instrumen

* extinction coefficient

sample

)

Measure using a Custom Factor

For example, for a sample measurement using an extinction coefficient of 55, the equation looks like this:

(550 AU * 55 ng-cm/μL) = 30,250 ng/μL

Note For measurements with 10 mm pathlength cuvettes, the upper absorbance limit is

1.5 AU, which is approximately 75 ng/μL for dsDNA.

Related Topics

• Detection Limits for All Applications

40 NanoDrop One User Guide Thermo Scientific

Measure Oligo DNA or Oligo RNA

Measures the concentration of purified ssDNA or RNA oligonucleotides that absorb at 260 nm.

Measure Oligo DNA or RNA

Reported Results

Settings

Detection Limits

Calculations

Measure Oligo DNA or Oligo RNA

Use the Oligo DNA and Oligo RNA applications to quantify oligonucleotides that absorb at 260 nm. Molar extinction coefficients are calculated automatically based on the user-defined base sequence of the sample. These applications report nucleic acid concentration and two absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction can also be used.

Note If the oligonucleotide has been modified, for example with a fluorophore dye, check with the oligo manufacturer to determine if the modification contributes absorbance at 260 nm. If it does, we recommend using the Microarray application to quantify nucleic acid concentration. The Microarray application includes a correction to remove any absorbance contribution due to the dye from the oligo quantification result.

Thermo Scientific NanoDrop One User Guide 41

Measure Oligo DNA or Oligo RNA

To measure Oligo DNA or Oligo RNA samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

42 NanoDrop One User Guide Thermo Scientific

 To measure an oligonucleotide sample

Example Oligo DNA spectrum

1. From the Home screen, select the Nucleic Acids tab and tap either Oligo DNA or Oligo RNA, as needed.

2. Specify the Oligo base sequence and a baseline

correction if desired.

3. Pipette 1–2 μL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette

light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

Measure Oligo DNA or Oligo RNA

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 1-2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if

Auto-Measure is off, lower arm and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Related Topics

• Best Practices for Nucleic Acid Measurements

Thermo Scientific NanoDrop One User Guide 43

Measure Oligo DNA or Oligo RNA

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

44 NanoDrop One User Guide Thermo Scientific

Oligo Reported Results

Nucleic acid concentration

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see

more/less sample data

UV spectrum

Purity ratios

Tap to select unitSample name;

tap to edit

Menu of options;

tap to open

Swipe screen left to

view table with more measurement results

Pinch and zoom to adjust axes; double-tap to reset

Tap to end experiment and export data

Oligo DNA measurement screen

For each measured sample, the Oligo DNA and Oligo RNA applications show the UV absorbance spectrum and a summary of the results. Here is an example:

Measure Oligo DNA or Oligo RNA

Measured oligo: TTT TTT TTT TTT TTT TTT TTT TTT

Note Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

Thermo Scientific NanoDrop One User Guide 45

Measure Oligo DNA or Oligo RNA

Oligo DNA and Oligo RNA reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

• sample details (application and sampling method used, i.e., pedestal or cuvette)

•sample name

• created on (date sample measurement was taken)

• nucleic acid concentration

• A260/A280

• A260/A230

•A260

•A280

•factor

• oligo sequence

• baseline correction

•stirrer status

Note The five nucleotides that comprise DNA and RNA exhibit widely varying A260/A280 ratios. See Oligo Purity Ratios for more information.

Related Topics

• Basic Instrument Operations

•Oligo Calculations

46 NanoDrop One User Guide Thermo Scientific

Settings for Oligo DNA and Oligo RNA Measurements

Add guanine

Add adenine

Add thymine (DNA) or uracil (RNA)

Add cytosine

Remove most recent base

The Oligo setup screen appears after you select the Oligo DNA or Oligo RNA application from the Nucleic Acids tab on the Home screen. To show the Oligo settings from the Oligo DNA or Oligo RNA measurement screen, tap > Oligo DNA Setup (or

Oligo RNA Setup).

Setting Available Options Description

Measure Oligo DNA or Oligo RNA

Oligo Base Sequence

for DNA: Use the G, A, T and C keys to specify the DNA base sequence

for RNA: Use the G, A, U and C keys to specify the RNA base sequence

Specify your DNA or RNA base sequence by tapping the corresponding keys:

Each time a base is added to the sequence, the software calculates the following:

• Factor. Constant used to calculate oligonucleotide concentration in modified

Beer’s Law equation. Based on extinction coefficient and pathlength:

f = 1/(



* b)

260

where: f= factor

 = molar extinction coefficient at 260 nm in ng-cm/μL

b = sample pathlength in cm (0.1 cm for nucleic acids measured with the NanoDrop One instrument)

Thermo Scientific NanoDrop One User Guide 47

Measure Oligo DNA or Oligo RNA

Setting Available Options Description

• Molecular Weight of oligo calculated from user-defined base sequence.

• Number of Bases entered.

• Molar Ext. Coefficient (260 nm). Molar extinction coefficient of oligo (in ng-cm/μL) at 260 nm calculated from entered base sequence.

• %GC. Percentage of guanine and cytosine residues in total number of bases entered.

Baseline Correction On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Corrects for any offset caused by light scattering particulates by subtracting measured absorbance at specified baseline correction wavelength from absorbance values at all wavelengths in sample spectrum. As a result, absorbance of sample spectrum is zero at specified baseline correction wavelength.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Baseline Correction.

Related Topics

• Instrument Settings

Detection Limits for Oligo DNA and Oligo RNA Measurements

The lower detection limits and reproducibility specifications for the oligonucleotide sample types (ssDNA and RNA) are provided here. The upper detection limits are dependent on the

upper absorbance limit of the instrument and the extinction coefficients for the user-defined base sequences.

To calculate upper detection limits for nucleic acid samples

To calculate upper detection limits in ng/μL, use the following equation:

(upper absorbance limit

For example, for a sample measurement using an extinction coefficient of 55, the equation looks like this:

(550 AU * 55 ng-cm/μL) = 30,250 ng/μL

Note For measurements with 10 mm pathlength cuvettes, the upper absorbance limit is

1.5 AU, which is approximately 75 ng/μL for dsDNA.

48 NanoDrop One User Guide Thermo Scientific

instrumen

* extinction coefficient

sample

)

Related Topics



260





N1–



–

N1–



• Detection Limits for All Applications

Calculations for Oligo DNA and Oligo RNA Measurements

Measure Oligo DNA or Oligo RNA

As with the other nucleic acid applications, the Oligo applications use the Beer-Lambert equation to correlate absorbance with concentration based on the sample’s extinction coefficient and pathlength. Because oligonucleotides are short, single-stranded molecules (or longer molecules of repeating sequences), their spectrum and extinction coefficient ( dependent on base composition and sequence.

(The generally accepted extinction coefficients and factors for single-stranded DNA and RNA provide a reasonable estimate for natural, essentially randomized, sequences but not for short, synthetic oligo sequences.) To ensure the most accurate results, we use the exact value of

The NanoDrop software allows you to specify the base sequence of an oligonucleotide before it is measured. For any entered base sequence, the software uses the equation at the right to calculate the extinction coefficient.

Tip: The extinction coefficient is wavelength specific for each oligonucleotide and can be affected by buffer type, ionic strength and pH.



to calculate oligonucleotide concentration.

260

) are closely

Extinction Coefficients for Oligonucleotides

The software uses the nearest neighbor method and the following formula to calculate molar extinction coefficients for specific oligonucleotide base sequences:

where:

 = molar extinction coefficient in L/mole-cm 

= 

nearest neighbor



= 

individual bases



= 

modifications, such as fluorescent dyes

Thermo Scientific NanoDrop One User Guide 49

Measure Oligo DNA or Oligo RNA

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Measured Values

A260 absorbance

Note: For micro-volume absorbance measurements and measurements

taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Nucleic acid absorbance values are measured at 260 nm using the normalized spectrum. This is the reported A260 value if Baseline Correction is not selected.

•If Baseline Correction is selected, the absorbance value at the correction wavelength is subtracted from the sample absorbance at 260 nm. The corrected absorbance at 260 nm is reported and used to calculate nucleic acid concentration.

A230, A280 absorbance

• Normalized absorbance values at 230 nm, 260 nm and 280 nm are used to calculate A260/A230 and A260/A280 ratios.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

•

50 NanoDrop One User Guide Thermo Scientific

Measure Oligo DNA or Oligo RNA

The five nucleotides that comprise DNA and RNA exhibit widely varying A260/A280 ratios. Estimated A260/A280 ratios for each independently measured nucleotide are provided below:

Guanine: 1.15 Adenine: 4.50 Cytosine: 1.51 Uracil: 4.00 Thymine: 1.47

The A260/A280 ratio for a specific nucleic acid sequence is approximately equal to the weighted average of the A260/A280 ratios for the four nucleotides present.

Note: RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of Thymine.

Related Topics

• Calculations for Nucleic Acid Measurements

Reported Values

• Nucleic acid concentration. Reported in selected unit (i.e., ng/μL, μg/uL or μg/mL). Calculations are based on modified Beer’s Law equation using corrected nucleic acid absorbance value.

• A260/A280 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm.

• A260/A230 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 230 nm.

Note: The traditional purity ratios (A260/A280 and A260/A230), which are used as indicators of the presence of various contaminants in nucleic acid samples, do not apply for oligonucleotides because the shapes of their spectra are highly dependent on their base compositions. See side bar for more information.

Thermo Scientific NanoDrop One User Guide 51

Measure Protein A280

Measures the concentration of purified protein populations that absorb at 280 nm.

Measure A280 Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Protein Concentration at A280

Use the Protein A280 application to quantify purified protein populations that contain amino acids such as tryptophan or tyrosine, or cys-cys disulfide bonds, which exhibit absorbance at 280 nm. This application reports protein concentration measured at 280 nm and one absorbance ratio (A260/A280). A single-point baseline correction can also be used. This application does not require a standard curve.

Note If your samples contain mainly peptide bonds and little or no amino acids, use the

Protein A205 application instead of Protein A280.

To measure Protein A280 samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop One User Guide 53

Measure Protein A280

Before you begin...

54 NanoDrop One User Guide Thermo Scientific

 To measure a Protein A280 sample

High concentration BSA sample

Low concentration BSA sample

1. From the Home screen, select the Proteins tab and tap Protein A280.

2. Specify a sample type and baseline correction if desired.

3. Pipette 1–2 μL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette

light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

Measure Protein A280

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if

Auto-Measure is off, lower arm and tap Measure.

– Cuvette: Tap Measure

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Thermo Scientific NanoDrop One User Guide 55

Measure Protein A280

Best practices for protein measurements

• Isolate and purify protein samples before measurement to remove impurities. Depending on the sample, impurities could include DNA, RNA and some buffer components. See

Preparing Samples for more information.

Note Extraction reagents that contribute absorbance between 200 nm and 280 nm will affect measurement results if present in samples (even residual amounts).

• Ensure the sample absorbance is within the instrument’s absorbance detection limits.

• Choosing a blank:

– For the Protein A280, Protein A205, and Proteins & Labels applications, blank with

the same buffer solution used to resuspend the analyte of interest. The blanking solution should be a similar pH and ionic strength as the analyte solution.

– For the Protein BCA, Protein Bradford, and Protein Lowry applications, blank with

deionized water (DI H

– For the Protein Pierce 660 application, blank with the reference solution used to

make the standard curve (reference solution should contain none of the standard protein stock). For more information, see Working with standard c u r ves.

O).

•Run a blanking cycle to assess the absorbance contribution of your buffer solution. If the buffer exhibits strong absorbance at or near the analysis wavelength (typically 280 nm or 205 nm), you may need to choose a different buffer or application, such as a colorimetric assay (for example, BCA or Pierce 660). See Choosing and Measuring a Blank for more information.

Note Buffers such as Triton X, RIPA, and NDSB contribute significant absorbance and are not compatible with direct A280 or A205 measurements.

• For micro-volume measurements:

– Ensure pedestal surfaces are properly cleaned and conditioned. (Proteins tend to stick

to pedestal surfaces.)

– Gently (but thoroughly) vortex samples before taking a measurement. Avoid

introducing bubbles when mixing and pipetting.

– Follow best practices for micro-volume measurements.

– Use a 2 μL sample volume. See Recommended Sample Volumes for more

information.

• For cuvette measurements (NanoDrop One

instruments only), use compatible cuvettes

and follow best practices for cuvette measurements.

56 NanoDrop One User Guide Thermo Scientific

Related Topics

• Best practices for protein measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Prepare Samples and Blanks

• Basic Instrument Operations

Protein A280 Reported Results

Protein A280 measurement screen

Measure Protein A280

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Thermo Scientific NanoDrop One User Guide 57

Measure Protein A280

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see

more/less sample data

Swipe screen left to view table with more measurement results

Pinch and zoom to adjust axes; double-tap to reset

Tap to end experiment and export data

UV spectrum

Tap to select unitMenu of options;

tap to open

Sample name; tap to edit

Protein concentration

Absorbance at 280 nm

Purity ratio

Note Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

Protein A280 reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

58 NanoDrop One User Guide Thermo Scientific

Measure Protein A280

Sample name;

tap to edit

Sampling method

Application

Date/time measured

Protein conc.

Absorbance at 280 nm

Sample type

Baseline correction wavelength

Baseline correction absorbance

Purity ratio

Settings for Protein A280 Measurements

Related Topics

• Basic Instrument Operations

• Protein A280 Calculations

To show the Protein A280 settings, from the Protein A280 measurement screen, tap >

Protein A280 Setup.

Protein A280 settings

The Protein A280 application provides a variety of sample type options for purified protein analysis.

Thermo Scientific NanoDrop One User Guide 59

Measure Protein A280

Each sample type applies a unique extinction coefficient to the protein calculations. If the extinction coefficient of the sample is known, choose the option and enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution. If you only need a rough estimate of protein concentration and the sample extinction coefficient is unknown, select the 1 Abs=1 mg/mL sample type option.

Tip Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

 + MW (molar) or 1% (mass)

Setting Available Options

Sample type

1 Abs = 1 mg/mL General reference Recommended when extinction coefficient is unknown

BSA 6.67 Calculates BSA (Bovine Serum Albumin) protein

IgG 13.7 Suitable for most mammalian antibodies (i.e.,

Mass Ext. Coefficient (L/gm-cm)

Description

and rough estimate of protein concentration is acceptable for a solution with no other interfering substances. Assumes 0.1% (1 mg/mL) protein solution produces 1.0A at 280 nm (where pathlength is 10 mm), i.e.,

1% = 10.

concentration using mass extinction coefficient (

6.67 L/gm-cm at 280 nm for 1% (i.e., 10 mg/mL) BSA solution. Assuming MW is 66,400 daltons (Da), molar extinction coefficient at 280 nm for BSA is approximately 43,824 M

immunoglobulin G or IgG). Calculates protein concentration using mass extinction coefficient (

13.7 L/gm-cm at 280 nm for 1% (i.e., 10 mg/mL) IgG solution. Assuming MW is 150,000 Da, molar extinction coefficient at 280 nm for IgG is approximately 210,000 M

-1cm-1

) of

Lysozyme 26.4 Calculates lysozyme protein concentration using mass

extinction coefficient ( for 1% (i.e., 10 mg/mL) lysozyme solution. Assumes molar extinction coefficient for egg white lysozyme ranges between 36,000 M

60 NanoDrop One User Guide Thermo Scientific

) of 26.4 L/gm-cm at 280 nm

-1cm-1

and 39,000 M-1cm-1.

Measure Protein A280

Setting Available Options

Other protein (

 + MW)

Other protein (

1%)

Baseline Correction

On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Mass Ext. Coefficient (L/gm-cm)

User entered molar extinction coefficient and molecular weight

Description

Assumes protein has known molar extinction coefficient () and molecular weight (MW), where:

(



)*10=(

molar

Enter MW in kiloDaltons (kDa) and molar extinction coefficient (

) in M

percent

)*(MW

-1cm-1

protein

divided by 1000 (i.e.,

)

/1000). For example, for protein with molar extinction

coefficient of 210,000 M

User entered mass extinction coefficient

N/A Corrects for any offset caused by light scattering

Assumes protein has known mass extinction coefficient (). Enter mass extinction coefficient in L/gm-cm for 10 mg/mL (

particulates by subtracting measured absorbance at specified baseline correction wavelength from absorbance values at all wavelengths in sample spectrum. As a result, absorbance of sample spectrum is zero at specified baseline correction wavelength.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Baseline Correction.

1%) protein solution.

-1cm-1

, enter 210.

To add or edit a custom protein, use Protein Editor.

Protein editor

Use the Protein Editor to add a custom protein to the list of available protein sample types in

Protein A280 Setup and Proteins & Labels Setup.

To access the Protein Editor:

• from the Home screen, tap > Protein Editor

• from the Protein A280 or Proteins & Labels measurement screen, tap >

Settings > Protein Editor

Thermo Scientific NanoDrop One User Guide 61

Measure Protein A280

Tap to add custom protein

Tap to edit selected custom protein

Tap to delete selected custom protein

Custom proteins (will appear in Sample Type list in Protein A280 Setup and Proteins & Labels Setup)

Tap to close Protein Editor

Protein Editor

These operations are available from the Protein Editor:

Add custom protein

– in Protein Editor, tap to show New Protein Type box

– enter unique Name for new protein (tap field to display keyboard, tap Done key to

close keyboard)

–enter Description for new protein

– specify whether to enter Molar Extinction coefficient or Mass Extinction coefficient

for custom protein

–if Mass Extinction coefficient is selected

– enter mass extinction coefficient in L/gm-cm for 10 mg/mL (

solution

1%) protein

62 NanoDrop One User Guide Thermo Scientific

Measure Protein A280

Tap a field to show keyboard; to close, tap Done key

–if Molar Extinction is selected

– enter molar extinction coefficient (

/1000). For example, for protein with molar extinction coefficient of

210,000 M

– enter molecular weight (MW) in kiloDaltons (kDa)

–tap OK to close New Protein Type box

After you choose OK, the new custom protein appears in the Type list in Protein A280 Setup and Proteins & Labels Setup.

Edit custom protein

– in Protein Editor, tap to select custom protein

– tap to show Edit Protein Type box

– edit any entries or settings

–tap OK

-1cm-1

, enter 210

) in M

-1cm-1

divided by 1000 (i.e.,

Thermo Scientific NanoDrop One User Guide 63

Measure Protein A280

Delete custom protein

– in Protein Editor, tap to select custom protein

–tap

Note Deleting a custom protein permanently removes the protein and all associated information from the software.

Related Topics

• Instrument Settings

Detection Limits for Protein A280 Measurements

Detection limits and reproducibility specifications for purified BSA proteins are provided

here. The BSA lower detection limit and reproducibility values apply to any protein sample

type. The upper detection limits are dependent on the upper absorbance limit of the instrument and the sample’s extinction coefficient.

To calculate upper detection limits for other (non-BSA) protein sample types

To calculate upper detection limits in ng/μL for proteins, use the following equation:

(upper absorbance limit

For example, if the sample’s mass extinction coefficient at 280 nm is 6.67 for a 1% (10 mg/mL) solution, the equation looks like this:

(550 / 6.67) * 10 = 824.6 (or ~825)

instrumen

Related Topics

• Detection Limits for All Applications

/mass extinction coefficient

sample

) * 10

64 NanoDrop One User Guide Thermo Scientific

Calculations for Protein A280 Measurements

Measure Protein A280

The Protein A280 application uses the Beer-Lambert

equation to correlate absorbance with concentration.

Solving Beer’s law for concentration yields the equation at the right.

The extinction coefficient of a peptide or protein is related to its tryptophan (W), tyrosin (Y) and cysteine (C) amino acid composition.

Tip: The extinction coefficient is wavelength specific for each protein and can be affected by buffer type, ionic strength and pH.

Beer-Lambert Equation (solved for concentration)

c = A / ( * b)

where:

A = UV absorbance in absorbance units (AU)

 = wavelength-dependent molar absorptivity coefficient (or extinction

coefficient) in liter/mol-cm

b = pathlength in cm

c = analyte concentration in moles/liter or molarity (M)

Note: Dividing the measured absorbance of a sample solution by its molar extinction coefficient yields the molar concentration of the sample. See

Published Extinction Coefficients for more information regarding molar

vs. mass concentration values.

Extinction Coefficients for Proteins

At 280 nm, the extinction coefficient is approximated by the weighted sum of the 280 nm molar extinction coefficients of the three constituent amino acids, as described in this equation:

 = (nW * 5500) + (nY * 1490) + (nC * 125)

where:

 = molar extinction coefficient

n = number of each amino acid residue 5500, 1490 and 125 = amino acid molar absorptivities at 280 nm

Thermo Scientific NanoDrop One User Guide 65

Measure Protein A280

This application offers six options (shown at right) for selecting an appropriate extinction coefficient for each measured sample, to be used in conjunction with Beer’s Law to calculate sample concentration.

If the extinction coefficient of the sample is known, choose the enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

Most sources report extinction coefficients for proteins measured at or near 280 nm in phosphate or other physiologic buffer. These values provide sufficient accuracy for routine assessments of protein concentration.

 + MW (molar) or 1% (mass) option and

Available Options for Extinction Coefficient

• 1 Abs = 1 mg/mL, where sample type and/or ext. coefficient is unknown (produces rough estimate of protein concentration)

• BSA (Bovine Serum Albumin, 6.67 L/gm-cm)

• IgG (any mammalian antibody, 13.7 L/gm-cm)

• Lysozyme (egg white lysozyme, 26.4 L/gm-cm)

• Other protein (

•

Note: See Sample Type for details.

Published Extinction Coefficients

Published extinction coefficients for proteins may be reported as:

• wavelength-dependent molar absorptivity (or extinction) coefficient

) with units of M

(

 + MW), user-specified molar ext. coefficient 1%), user-specified mass ext. coefficient

-1cm-1

The equation at the right shows the relationship between molar extinction coefficient ( percent extinction coefficient (

1%).



molar

) and

• percent solution extinction coefficient (1%) with units of (g/100 mL) 1 cm cuvette)

• protein absorbance values for 0.1% (i.e., 1 mg/mL) solutions

Tip: Assess published values carefully to ensure unit of measure is applied correctly.

Conversions Between 

(

molar

Example: To determine percent solution extinction coefficient (1%) for a protein that has a molar extinction coefficient of 43,824 M molecular weight (MW) of 66,400 daltons (Da), rearrange and solve the above equation as follows:

1% = (

-1cm-1

(i.e., 1% or 1 g/100 mL solution measured in a

and 1%

molar

) * 10 = (1%) * (MW

* 10) / (MW

molar

protein

)

-1cm-1

and a

1% = (43,824 * 10) / 66,400 Da) 1% = 6.6 g/100 mL

66 NanoDrop One User Guide Thermo Scientific

Measure Protein A280

To determine concentration (c) of a sample in mg/mL, use the equation at the right and a conversion factor of 10.

Tip: The NanoDrop One software includes the conversion factor when reporting protein concentrations.

Calculated protein concentrations are based on the absorbance value at 280 nm, the selected (or entered) extinction coefficient and the sample pathlength. A single-point baseline correction (or analysis correction) may be applied.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Absorbance values at 260 nm and 280 nm are used to calculate purity ratios for the measured protein samples.

Purity ratios are sensitive to the presence of contaminants in the sample, such as residual solvents and reagents typically used during sample purification.

Conversions Between g/100 mL and mg/mL

Example: If measured absorbance for a protein sample at 280 nm relative to the reference is 5.8 A, protein concentration can be calculated as:

Measured Values

A280 absorbance

Note: For micro-volume absorbance measurements and measurements

taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Protein absorbance values are measured at 280 nm using the normalized spectrum. If Baseline Correction is not selected, this is the reported A280 value and the value used to calculate protein concentration.

•If Baseline Correction is selected, the normalized and baseline-corrected absorbance value at 280 nm is reported and used to calculate protein concentration.

A260 absorbance

in mg/mL = (A / 1%) * 10

protein

= (A / 1%) * 10

protein

= (5.8/6.6 g/100 mL) * 10

protein

= 8.79 mg/mL

protein

• Normalized and baseline-corrected (if selected) absorbance value at 260 nm is also reported.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

•

Thermo Scientific NanoDrop One User Guide 67

Measure Protein A280

Reported Values

• Protein concentration. Reported in selected unit (mg/mL or μg/mL). Calculations are based on Beer-Lambert equation using corrected protein absorbance value.

• A260/A280 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm. An A260/A280 purity ratio of ~0.57 is generally accepted as “pure” for proteins.

Note: Although purity ratios are important indicators of sample quality, the best indicator of protein quality is functionality in the downstream application of interest (e.g., real-time PCR).

68 NanoDrop One User Guide Thermo Scientific

Measure Proteins and Labels

Measures the concentration of purified proteins that have been labeled with up to two fluorescent dyes.

Measure Labeled Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Labeled Protein Samples

Use the Proteins and Labels application to quantify proteins and fluorescent dyes for protein array conjugates, as well as metalloproteins such as hemoglobin, using wavelength ratios. This application reports protein concentration measured at 280 nm, an A269/A280 absorbance ratio, and the concentrations and measured absorbance values of the dyes, allowing detection of dye concentrations as low as 0.2 picomole per microliter. This information is useful for evaluating protein/dye conjugation (degree of labeling) for use in downstream applications.

To measure labeled protein samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop One User Guide 69

Measure Proteins and Labels

Before you begin...

70 NanoDrop One User Guide Thermo Scientific

 To measure a labeled protein sample

Dye absorbance peak used to calculate dye concentration

A280 absorbance peak used to calculate protein concentration

Peptide backbone

Typical sample spectrum measured with Proteins & Labels application

1. From the Home screen, select the Proteins tab and then tap Protein & Labels.

2. Specify the sample type and the type of dye(s) used.

Tip: Select a dye from the pre-defined list or add a custom dye using the Dye/Chromophore Editor.

3. Pipette 1–2 μL of the blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette

light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Measure Proteins and Labels

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if

– Cuvette: Tap Measure

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Auto-Measure is off, lower arm and tap Measure.

Thermo Scientific NanoDrop One User Guide 71

Measure Proteins and Labels

Related Topics

• Best practices for protein measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Prepare Samples and Blanks

• Basic Instrument Operations

Proteins & Labels Reported Results

Proteins & Labels measurement screen

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

72 NanoDrop One User Guide Thermo Scientific

Measure Proteins and Labels

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see

more/less sample data

UV-visible spectrum

Tap to select unitMenu of options;

tap to open

Sample name; tap to edit

Protein concentration

Dye concentration(s)

Swipe screen left to view table with more measurement results

Pinch and zoom to adjust axes; double-tap to reset

Tap to end experiment and export data

Note

• A baseline correction is performed at 750 nm (absorbance value at 750 nm is subtracted from absorbance values at all wavelengths in sample spectrum).

• Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

Thermo Scientific NanoDrop One User Guide 73

Measure Proteins and Labels

Proteins & Labels reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

Reported values for Proteins & Labels application

• Sample details (application and sampling method used, i.e., pedestal or cuvette)

•Sample Name

•Creation date

•Protein

•A280

•Sample Type

•Dye 1/Dye 2

• Sloping Dye Correction

•Analysis Correction

Related Topics

• Basic Instrument Operations

• Proteins & Labels calculations

Settings for Proteins and Labels Measurements

To show the Proteins & Labels settings, from the Proteins & Labels measurement screen, tap

> Proteins & Labels Setup.

74 NanoDrop One User Guide Thermo Scientific

Measure Proteins and Labels

Setting Available Options

Sample type

1 Abs = 1 mg/mL

BSA

IgG

Lysozyme

Other protein

 + MW)

(

Other protein (

1%)

Analysis Correction

On or off

Enter analysis correction wavelength in nm or use default value (340 nm)

Mass Ext. Coefficient (L/gm-cm)

General reference

6.67

Description

Tap he re for detailed description of each available setting.

Each sample type applies a unique extinction coefficient to the protein calculations. If the extinction coefficient of the

13.7

26.4

user-entered molar extinction coefficient/molecular weight

User entered mass extinction coefficient

sample is known, choose the

 + MW (molar) or 1%

(mass) option and enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution. If you only need a rough estimate of protein concentration and the sample extinction coefficient is unknown, select the 1 Abs=1 mg/mL sample type option.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

N/A Corrects sample absorbance measurement for any offset

caused by light scattering particulates by subtracting absorbance value at specified analysis correction wavelength from absorbance value at analysis wavelength. Corrected value is used to calculate sample concentration.

Dye 1/Dye 2

Ty pe

Cy3, 5, 3.5, or 5.5, Alexa Fluor 488, 546, 555, 594, 647, or 660

See

Dye/Chromophore Editor for specific

values for each dye

Dye 1/Dye 2 Unit

picomoles/microliter (pmol/uL), micromoles

not applicable Select unit for reporting dye concentrations.

(uM), or millimoles (mM)

Sloping Dye Correction

To add or edit a custom protein, use Protein Editor.

Analysis Correction affects calculation for protein concentration only.

To add custom dye or edit list of available dyes, use Dye/Chromophore Editor.

Sloping Dye Correction affects calculations for dye concentration only.

On or off Corrects dye absorbance measurements for any offset

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Analysis Correction.

Select pre-defined dye used to label sample material, or one that has been added using Dye/Chrom. Editor.

caused by light scattering particulates by subtracting absorbance value of a sloping baseline from 400 nm to 750 nm from absorbance value at dye’s analysis wavelength.

Thermo Scientific NanoDrop One User Guide 75

Measure Proteins and Labels

Related Topics

• Instrument Settings

• Protein Editor

• Dye/Chromophore Editor

Detection Limits for Proteins and Labels Measurements

Detection limits and reproducibility specifications for purified BSA proteins and dyes that are pre-defined in the software are provided here. The BSA lower detection limit and reproducibility values apply to any protein sample type. The upper detection limits are dependent on the upper absorbance limit of the instrument and the sample’s extinction coefficient.

To calculate upper detection limits for other (non-BSA) protein sample types

To calculate upper detection limits in ng/μL for proteins, use the following equation:

(upper absorbance limit

For example, if the sample’s mass extinction coefficient at 280 nm is 6.67 for a 1% (10 mg/mL) solution, the equation looks like this:

(550 / 6.67) * 10 = 824.6 (or ~825)

instrumen

Related Topics

• Detection Limits for All Applications

/mass extinction coefficient

sample

) * 10

76 NanoDrop One User Guide Thermo Scientific

Calculations for Proteins and Labels Measurements

Measure Proteins and Labels

As with the other protein applications, Proteins & Labels uses the Beer-Lambert equation to correlate absorbance with concentration based on the sample’s extinction coefficient and pathlength.

If the extinction coefficient of the sample is known, choose the enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

 + MW (molar) or 1% (mass) option and

Available Options for Extinction Coefficient

• 1 Abs = 1 mg/mL, where sample type and/or ext. coefficient is unknown (produces rough estimate of protein concentration)

• BSA (Bovine Serum Albumin, 6.67 L/gm-cm)

• IgG (any mammalian antibody, 13.7 L/gm-cm)

• Lysozyme (egg white lysozyme, 26.4 L/gm-cm)

• Other protein (

 + MW), user-specified molar ext. coefficient 1%), user-specified mass ext. coefficient

Note: See Sample Type for details.

Measured Values

A280 absorbance

Note: The absorbance value at 750 nm is subtracted from all wavelengths

• Protein absorbance values are measured at 280 nm using the 750 nm-corrected and normalized spectrum. If Analysis Correction and Dye Correction are not selected, this is the reported A280 value and the value used to calculate protein concentration.

•If Analysis Correction is selected, the 750-corrected, normalized and analysis-corrected absorbance value at 280 nm is reported and used to calculate protein concentration.

• If a Dye is used, the 750-corrected, normalized, analysis-corrected and

dye-corrected absorbance value at 280 nm is reported and used to

calculate protein concentration.

Thermo Scientific NanoDrop One User Guide 77

Measure Proteins and Labels

Dye absorbance

• Dye absorbance values are measured at specific wavelengths. See

Dye/Chromophore Editor for analysis wavelengths used.

Dye correction

• Pre-defined dyes have known correction values for A260 and A280. See Dye/Chromophore Editor for correction values used.

• A280 dye correction is subtracted from A280 absorbance value used to calculate protein concentration.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

Related Topics

• Beer-Lambert Equation

• Protein A280 Calculations

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

Reported Values

• Protein concentration. Reported in selected unit (mg/mL or μg/mL). Calculations are based on Beer-Lambert equation using corrected protein absorbance value.

• Dye1/Dye2 concentration. Reported in pmol/μL. Calculations are based on Beer’s Law equation using (sloping) baseline-corrected dye absorbance value(s).

78 NanoDrop One User Guide Thermo Scientific

Measure Protein A205

Measures the concentration of purified protein populations that absorb at 205 nm.

Measure A205 Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Protein Concentration at A205

Use the Protein A205 application to quantify purified peptides and other proteins that contain peptide bonds, which exhibit absorbance at 205 nm. This application reports protein concentration and two absorbance values (A205 and A280). A single-point baseline correction can also be used. This application does not require a standard curve.

Note If your samples contain mainly amino acids such as tryptophan or tyrosine, or cys-cys disulfide bonds, use the Protein A280 application instead of Protein A205.

To measure Protein A205 samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop One User Guide 79

Measure Protein A205

Before you begin...

 To measure a Protein A205 sample

1. From the Home screen, select the Proteins tab and then tap Protein A205.

2. Specify a sample type and baseline correction if desired.

3. Pipette 1–2 μL of the blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 2 μL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

–Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm and

tap Measure.

– Cuvette: Tap Measure

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Related Topics

• Best Practices for Protein Measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

80 NanoDrop One User Guide Thermo Scientific

• Prepare Samples and Blanks

• Basic Instrument Operations

Measure Protein A205

Thermo Scientific NanoDrop One User Guide 81

Measure Protein A205

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see

more/less sample data

Swipe screen left to view table with more measurement results

Pinch and zoom to adjust axes; double-tap to reset

Tap to end experiment and export data

UV spectrum

Tap to select unitMenu of options;

tap to open

Sample name; tap to edit

Protein concentration

Absorbance at 205 nm

Absorbance at 280 nm

Protein A205 Reported Results

Protein A205 measurement screen

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Note Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

82 NanoDrop One User Guide Thermo Scientific

Measure Protein A205

Sample name;

tap to edit

Sampling method

Application

Date/time measured

Protein conc.

Absorbance at 205 nm

Sample type

Baseline Correction wavelength

Baseline Correction absorbance

Absorbance at 280 nm

Protein A205 reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

Settings for Protein A205 Measurements

Thermo Scientific NanoDrop One User Guide 83

Related Topics

• Basic Instrument Operations

• Protein A205 Calculations

To show the Protein A205 settings, from the Protein A205 measurement screen, tap >

Protein A205 Setup.

Measure Protein A205

Setting Available Options

Sample type 31 31 Assumes 0.1% (1 mg/mL) at 205 nm = 31

Scopes 27 + 120 * (A280/A205) Assumes

Other protein

1%)

(

Baseline Correction

On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Mass Ext. Coefficient (L/gm-cm)

Description

0.1% (1 mg/mL) at 205 nm = 27 + 120 *

(A280/A205)

User entered mass extinction coefficient

N/A Corrects for any offset caused by light scattering

Assumes protein has known mass extinction coefficient

(). Enter mass extinction coefficient in L/gm-cm for

1mg/mL (

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Baseline Correction.

0.1%) protein solution.

Related Topics

• Instrument Settings

84 NanoDrop One User Guide Thermo Scientific

Calculations for Protein A205 Measurements

Measure Protein A205

As with the other protein applications, Proteins A205 uses the Beer-Lambert equation to correlate absorbance with concentration based on the sample’s extinction coefficient and pathlength.

This application offers three options (shown at right) for selecting an appropriate extinction coefficient for each measured sample, to be used in conjunction with Beer’s Law to calculate sample concentration.

If the extinction coefficient of the sample is known, choose the Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

Calculated protein concentrations are based on the absorbance value at 205 nm, the selected (or entered) extinction coefficient and the sample pathlength. A single-point baseline correction may also be applied.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on the sample sequence.

1% (mass) option and enter the value.

Available Options for Extinction Coefficient

• 31, assumes

• Scopes, assumes

0.1% (1 mg/mL) at 205 nm = 31

0.1% (1 mg/mL) at 205 nm = 27 + 120 *

(A280/A205)

• Other protein, enter mass extinction coefficient in L/gm-cm for

1mg/mL (0.1%) protein solution

Note: See Sample Type for details.

Measured Values

A205 absorbance

Note: For micro-volume absorbance measurements and measurements

taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Protein absorbance values are measured at 205 nm using the normalized spectrum. If Baseline Correction is not selected, this is the reported A205 value and the value used to calculate protein concentration.

•If Baseline Correction is selected, the normalized and baseline-corrected absorbance value at 205 nm is reported and used to calculate protein concentration.

A280 absorbance

• Normalized and baseline-corrected (if selected) absorbance value at 280 nm is also reported.

Thermo Scientific NanoDrop One User Guide 85

Measure Protein A205

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

Reported Values

• Protein concentration. Reported in selected unit (mg/mL or μg/mL). Calculations are based on Beer-Lambert equation using corrected protein absorbance value.

Related Topics

• Beer-Lambert Equation

• Protein A280 Calculations

86 NanoDrop One User Guide Thermo Scientific

Measure Protein BCA

Measures total protein concentration of unpurified protein samples using a bicinchoninic acid colorimetric detection reagent.

Measure Total Protein

Reported Results

Settings

Detection Limits

Measure Total Protein Concentration

The Protein BCA assay uses bicinchoninic acid as a colorimetric detection reagent to determine total protein concentration in unpurified protein samples. This application is useful for measuring dilute protein solutions or proteins in the presence of components that exhibit significant absorbance between 200 nm and 280 nm, which rules out direct protein measurements at 280 nm or 205 nm. This application measures absorbance at 562 nm and uses a standard curve to calculate protein concentration. A single-point baseline correction is applied.

Theory of Protein BCA assay

The Protein BCA assay uses bicinchoninic acid (BCA) as the detection reagent for Cu+1, which is formed when Cu purple reaction product is formed by the chelation of two molecules of BCA with one cuprous ion (Cu 562 nm and baseline-corrected using the absorbance value at 750 nm. Pre-formulated kits of BCA reagent and CuSO

Thermo Scientific NanoDrop One User Guide 87

). The resulting Cu-BCA chelate formed in the presence of protein is measured at

is reduced by certain proteins in an alkaline environment. A

are available from us or a local distributor.

Measure Protein BCA

Protein assay kits and protocols

Please refer to the NanoDrop website for up-to-date kits and protocols for the NanoDrop One instruments. Follow the assay kit manufacturer’s recommendations for all standards and samples (unknowns). Ensure each is subjected to the same timing and temperature throughout the assay.

Protein standards for generating a standard curve may also be provided by the kit manufacturer. Since the NanoDrop One pedestals can measure higher protein concentrations than traditional cuvette-based spectrophotometers, you may need to supply your own protein standards at higher concentrations than provided by the manufacturer. For example, additional standards may be required to ensure the standard curve covers the dynamic range of the assay and the expected range of the unknown samples.

Working with standard curves

A standard curve is required for colorimetric protein analysis.

• Each experiment requires a new standard curve.

• Prepare standards and unknown samples the same way. See the kit manufacturer’s guidelines and recommendations.

– All reference and standards solutions should be the same buffer used to resuspend

the samples plus the same volume of reagent added to the samples.

– First standard is a reference measurement. The reference solution should contain

none of the analyte of interest. (The reference measurement is not the same as a blank measurement. This application requires both.)

– Concentration range of the standards must cover the dynamic range of the assay

and the expected range of the unknown samples. Sample analyte concentrations are not extrapolated beyond the concentration of the highest standard.

• Use the application setup screen to enter concentration values for the standards and to specify how standards and samples will be measured (number of replicates, etc.).

– Depending on the Curve Type setting, a standard curve can be generated using two

or more standards.

–The software requires one reference measurement and allows up to 7 standards.

– Concentration values for standards can be entered in any order but the standards

must be measured in the order in which they were entered; however, best practice dictates that standards be measured from the lowest concentration of the standard analyte stock to the highest.

88 NanoDrop One User Guide Thermo Scientific

Measure Protein BCA

Reference concentration and absorbance value

Standard concentrations and absorbance values

Swipe left one screen

to view standard curve

Menu; tap to open

Press and hold any row to view details

• For all colorimetric assays except Protein Pierce 660, blank the instrument with DI H

O (deionized water). For Protein Pierce 660, blank with the reference solution (see

below).

• Measure the reference and all standards before you start analyzing samples. (After the first sample has been measured, no additional changes are allowed to the standard curve.)

As you measure the standards, a measurement screen appears, similar to the measurement screens for samples.

Thermo Scientific NanoDrop One User Guide 89

Measure Protein BCA

Swipe left one screen to view data table for standards

Curve type setting

R2 value (1.0 equals perfect fit)

White circles indicate data points for standards

Standard curve

Press and hold any row to view details

Swipe left one screen to see the standard curve as you build it. Here is an example:

The R perfect fit; all points lie exactly on the curve).

value indicates how well the standard curve fits the standard data points (1.0 is a

90 NanoDrop One User Guide Thermo Scientific

Measure Protein BCA

Press and hold any row to view details

Tap to delete

this measurement

Swipe left one screen to see the data table for the standards. Here is an example:

Press and hold a row in any of the previous screens to view details about an individual

standard. Here is an example:

Thermo Scientific NanoDrop One User Guide 91

Measure Protein BCA

After the minimum number of standards has been measured for the selected curve type, a message similar to the following appears:

Load more standards: returns to the setup screen where you can add or edit the concentration value for any standard and then measure the standard.

Run samples: continues to sample measurement screen, after which standards can no longer be edited.

• You can add, edit or delete a standard any time before the first sample measurement.

Add standard

– from standards measurement screen, tap > [application name] Setup

– tap the next empty Concentration field and enter the concentration value for the new

standard

–tap Done

Edit standard

– from standards measurement screen, tap > [application name] Setup

– tap the Concentration field and edit the concentration value

–tap Done

Delete standard

– from standards measurement screen, standard curve screen, or standards data table,

press and hold the row to show Standard Details box

–tap

The standard no longer appears in the table on the measurement screen and its concentration value no longer appears on the setup screen.

Note You can use this method to delete the reference measurement; however, a new reference must be measured immediately afterwards.

92 NanoDrop One User Guide Thermo Scientific

• After the minimum number of standards has been measured for the selected curve type, the message “Invalid Curve” changes to “Valid Curve.” (This occurs even when additional standards have been defined but not yet measured.) If the “Invalid Curve” message remains after all entered standards have been measured, try:

– selecting a different curve type

– remeasuring standards using the correct standard material

Valid Curve indicator: This is only an indicator that the required minimum number of points has been established for the selected curve type. It does not validate the integrity of the curve. For example, additional standards may be required to cover the expected assay concentration range.

To measure Protein BCA standards and samples

Measure Protein BCA

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

 To measure Protein BCA standards and samples

1. From the Home screen, select the Proteins tab and tap Protein BCA.

2. Specify a curve type and number of replicates for each standard and enter the

concentration of each standard.

Tip: For this assay, we recommend setting Curve Type to “Linear”.

3. Measure blank:

– pipette 2 μL DI H

O onto lower pedestal and lower arm, or insert DI H2O

blanking cuvette into cuvette holder

Thermo Scientific NanoDrop One User Guide 93

Measure Protein BCA

Tip: If using a cuvette, make sure to align cuvette light path with instrument light path.

–tap Blank and wait for measurement to complete

– lift arm and clean both pedestals with new laboratory wipe, or remove cuvette

4. Measure reference standard:

– pipette 2 μL reference solution onto pedestal, or insert reference cuvette (reference

solution should contain none of the standard protein stock, see Working With

Standard Curves for details)

– lower arm to start measurement (or tap Measure if Auto-Measure is off)

– lift arm and clean both pedestals with new wipe, or remove cuvette

– if Replicates setting is greater than 1, repeat measurement

5. Measure remaining standards:

– pipette 2 μL standard 1 onto pedestal, or insert standard 1 cuvette

– lower arm to start measurement (or tap Measure if Auto-Measure is off)

– lift arm and clean both pedestals with new wipe, or remove cuvette

– if Replicates setting is greater than 1, repeat measurement

– repeat substeps above for each additional standard (when specified number of

standards and replicates have been measured, a message asks whether to load more standards or begin measuring samples)

– if finished measuring standards, tap Done (swipe left to view standard curve)

6. Measure samples:

– pipette 2 μL sample 1 onto pedestal, or insert sample 1 cuvette

– lower arm to start measurement (or tap Measure if Auto-Measure is off)

– lift arm and clean both pedestals with new wipe, or remove cuvette

– if Replicates setting is greater than 1, repeat measurement

7. When you are finished measuring samples, tap End Experiment.

8. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Related Topics

• Best practices for protein measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

94 NanoDrop One User Guide Thermo Scientific

Thermo NanoDrop One User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Contents

About the NanoDrop One Spectrophotometer

Instrument Models and Features

Optional Accessories

Register Your Instrument

Update Software

Applications

Detection Limits for All Applications

Measure dsDNA, ssDNA or RNA

Measure dsDNA, ssDNA or RNA

Nucleic Acid Reported Results

Setting for Nucleic Acid Measurements

Calculations for Nucleic Acid Measurements

Measure Microarray

Measure Microarray Samples

Microarray Reported Results

Settings for Microarray Measurements

Calculations for Microarray Measurements

Measure using a Custom Factor

Measure Nucleic Acid using a Custom Factor

Custom Factor Reported Results

Settings for Nucleic Acid Measurements using a Custom Factor

Detection Limits for Nucleic Acid Measurements using a Custom Factor

Measure Oligo DNA or Oligo RNA

Measure Oligo DNA or Oligo RNA

Oligo Reported Results

Settings for Oligo DNA and Oligo RNA Measurements

Detection Limits for Oligo DNA and Oligo RNA Measurements

Calculations for Oligo DNA and Oligo RNA Measurements

Measure Protein A280

Measure Protein Concentration at A280

Protein A280 Reported Results

Settings for Protein A280 Measurements

Detection Limits for Protein A280 Measurements

Calculations for Protein A280 Measurements

Measure Proteins and Labels

Measure Labeled Protein Samples

Proteins & Labels Reported Results

Settings for Proteins and Labels Measurements

Detection Limits for Proteins and Labels Measurements

Calculations for Proteins and Labels Measurements

Measure Protein A205

Measure Protein Concentration at A205

Protein A205 Reported Results

Settings for Protein A205 Measurements

Calculations for Protein A205 Measurements

Measure Protein BCA

Measure Total Protein Concentration