Bio-Rad DC Protein Assay User Manual
DC Protein Assay
Instruction
Manual
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Table of Contents
Section 1 Introduction and Principle ……………………………… 1
Section 2 Product Description ……………………………………… 1
Section 3 Materials Required but Not Supplied…………………… 1
3.1 Safety Considerations …………………………………………… 2
Section 4 Reagent Compatibility …………………………………… 2
Section 5 Instructions ……………………………………………… 3
5.1 Standard Assay Protocol ………………………………………… 3
5.2 Microplate Assay Protocol ……………………………………… 3
Section 6 Response of Various Proteins …………………………… 4
Section 7 Storage …………………………………………………… 4
Section 8 Troubleshooting Guide…………………………………… 5
Section 9 Ordering Information …………………………………… 6
Section 10 Related Materials ………………………………………… 6
Section 11 References ………………………………………………… 7
Section 12 Safety Information ……………………………………… 8
Section 1
Introduction and Principle
The Bio-Rad DC Protein Assay is a colorimetric assay for protein concen-
tration following detergent solubilization. The reaction is similar to the well-documented Lowry1assay, but with the following improvements: The reaction
reaches 90% of its maximum color development within 15 minutes thereby
saving valuable time, and the color changes not more than 5% in 1 hour or 10%
in 2 hours after the addition of reagents.
The assay is based on the reaction of protein with an alkaline copper tartrate
solution and Folin reagent. As with the Lowry assay, there are two steps which
lead to color development: The reaction between protein and copper in an alkaline
medium, and the subsequent reduction of Folin reagent by the copper-treated protein.1Color development is primarily due to the amino acids tyrosine and tryptophan, and to a lesser extent, cystine, cysteine, and histidine.
reduction of the Folin reagent by loss of 1, 2, or 3 oxygen atoms, thereby producing one or more of several possible reduced species which have a characteristic blue
color with maximum absorbance at 750 nm and minimum absorbance at 405 nm.
Section 2
Product Description
Reagent package (catalog number 500-0116) includes:
250 ml REAGENT A, an alkaline copper tartrate solution
2000 ml REAGENT B, a dilute Folin Reagent
5 ml REAGENT S
(Sufficient for 500 standard assays or 10,000 microplate assays)
1,2
Proteins effect a
2
The reagent package may be purchased as a kit with a bovine gamma globulin standard (kit catalog number 500-011 1) or bovine serum albumin standard
(kit catalog number 500-0112).
Section 3
Materials Required but Not Supplied
For standard assay:
13 x 100 mm test tubes
Reservoir for working reagent (size depends on amount of reagent that
will be prepared)
Pipets accurately delivering 100 µl, 500 µl, and 4.0 ml
Graduated cylinders or pipets for reagent preparation
Spectrophotometer set to 750 nm
1
Vortex mixer
Plastic or glass cuvettes with 1 cm path length matched to laboratory spec-
trophotometer
Test tube rack to hold 13 x 100 mm test tubes
For microplate assay:
Microtiter plates
Reservoir for working reagent
Pipets for reagent preparation
Pipets accurately delivering 5 µl, 25 µl, and 200 µl
Microplate reader set to 750 nm
3.1 Safety Considerations
Eye protection and gloves should be worn while using this product. Consult
MSDS at the end of this manual for additional information.
Section 4
Reagent Compatibility
The listed compounds were tested and found to be compatible with the BioRad DC Protein Assay. In some cases, the presence of one or more of these
substances will effect a change in the response of the protein to the assay
reagents; therefore, the standard should always be prepared in the same buffer
as the sample.
10% SDS 1% CHAPS 2% NP-40
1% Triton†X-100 1% CHAPSO 1% Thesit
1% Tween†20 1% Octyl glucoside 1% Brij†-35
0.2% C12E8* 0.1 M Tris, pH 8 0.5 M NaOH
0.5 M HCl 0.5 M (NH4)2SO
0.05 M CaCl
0.05% Sodium azide 1 mM DTT (dithiothreitol)
2
4
0.025 M EDTA
0.4 M Guanidine HCl 4 M Urea
†
Note: The DC Protein Assay is incompatible with 2-mercaptoethanol (BME)
† BRIJ and TWEEN are registered trademarks of Atlas Chemical. THESIT is
a registered trademark of Desitin Arzneimittel GMBH. TRITON is a registered
trademark of Rohm and Haas.
*octaethyleneglycol dodecyl ether
2
Section 5
Instructions
5.1 Standard Assay Protocol
1. Preparation of working reagent
Add 20 µl of reagent S to each ml of reagent A that will be needed for the run.
(This working reagent A' is stable for one week even though a precipitate
will form after one day. If precipitate forms, warm the solution and vortex.
Do not pipet the undissolved precipitate, as this will likely plug the tip of the
pipet, thereby altering the volume of reagent that is added to the sample.)
If samples do not contain detergent, you may omit step #1 and simply use
reagent A as supplied.
2. Prepare 3 - 5 dilutions of a protein standard containing from 0.2 mg/ml to
about 1.5 mg/ml protein. A standard curve should be prepared each time the
assay is performed. For best results, the standards should always be prepared
in the same buffer as the sample.
3. Pipet 100 µl of standards and samples into clean, dry test tubes.
4. Add 500 µl of reagent A' or A (see note from step 1) into each test tube.
Vortex.
5. Add 4.0 ml reagent B into each test tube and vortex immediately.
6. After 15 minutes, absorbances can be read at 750 nm. The absorbances will
be stable at least 1 hour. (See Troubleshooting Guide for recommendation
on using a wavelength other than 750 nm.)
5.2 Microplate Assay Protocol
1. Preparation of working reagent
Add 20 µl of reagent S to each ml of reagent A that will be needed for the run.
(This working reagent A' is stable for 1 week even though a precipitate
will form after 1 day. If precipitate forms, warm the solution and vortex. Do
not pipet the undissolved precipitate, as this will likely plug the tip of the pipet,
thereby altering the volume of reagent that is added to the sample.)
If samples do not contain detergent, you may omit step #1 and simply use
reagent A as supplied.
2. Prepare 3 - 5 dilutions of a protein standard containing from 0.2 mg/ml to about
1.5 mg/ml protein. A standard curve should be prepared each time the assay
is performed. For best results, the standar d should be prepar ed in the same
buffer as the sample.
3. Pipet 5 µl of standards and samples into a clean, dry microtiter plate.
3
4. Add 25 µl of reagent A' or reagent A (see note from step 1) into each well.
protein concentration, mg/ml
absorbance
BSA
IgG
Ribonuclease A
Ovalbumin
Conalbumin
2.01.00.0
5. Add 200 µl reagent B into each well. If microplate reader has a mixing func-
tion available, place plate in reader and let the plate mix for 5 seconds. If not,
gently agitate the plate to mix the reagents. If bubbles form, pop them with a
clean, dry pipet tip. Be careful to avoid cross-contamination of sample wells.
6. After 15 minutes, absorbances can be read at 750 nm. The absorbances will
be stable for about 1 hour. (See Troubleshooting Guide for recommendation
on using a wavelength other than 750 nm.)
Section 6
Response of Various Proteins
As with any colorimetric assay , different proteins will elicit greater or less er color formation. The following proteins have been assayed with the protein
assay. As demonstrated by the graph, there is a slight variation in color development with different proteins.
Section 7
Storage
Lyophilized preparations of Protein Standard I (bovine gamma globulin) and
Protein Standard II (bovine serum albumin), if included, should be refrigerated upon
arrival. These lyophilized preparations have a shelf life of one year at 4 °C. Rehydrated
and stored at 4 °C, the protein solutions should be used within 60 days. Rehydrated
and stored at -20 °C, the protein solutions should be used within 6 months.
REAGENT A, REAGENT B, and REAGENT S should be stored away from
direct sunlight at room temperature (25-30 °C). (Reagents A and B may also be
stored in the refrigerator.) All reagents are good for 6 months from date of purchase.
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