Bio-Rad Experion Protein Analysis Kits User Manual

Experion™
Pro260 Analysis Kit

Instruction Manual

Catalog #700-7101

Bio-Rad Technical Support

For help and advice regarding products from the Experion™ automated electrophoresis system, please contact the Bio-Rad

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or use of this information.

Bio-Lyte, Criterion, Experion, Precision Plus Protein, and ReadyPrep are trademarks of Bio-Rad Laboratories. B-PER is a

trademark of Pierce. BugBuster is a trademark of Novagen. Coomassie and Pluronic are trademarks of BASF Aktiengesellschaft.

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Zwittergent is a trademark of EMD Chemicals Inc.

The dyes in Experion kits are manufactured by Molecular Probes, Inc. and are licensed for research use only.

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licensed by Caliper Life Sciences, Inc. to sell products using the LabChip technology for research use only.

These products are licensed under U.S. patents 5,863,753; 5,658,751; 5,436,134; and 5,582,977, and pending

patent applications, and related foreign patents, for internal research and development use only in detecting,

quantitating, and sizing macromolecules, in combination with microfluidics, where internal research and development use

expressly excludes the use of this product for providing medical, diagnostic, or any other testing, analysis, or screening services,

or providing clinical information or clinical analysis, in any event in return for compensation by an unrelated party.

Copyright © 2010 Bio-Rad Laboratories, Inc.

Contents

Chapter 1: Experion™ Pro260 Analysis Kit ............................................. 1

1.1 Product Description .......................................................... 2

1.2 Kit Components ............................................................. 3

1.3 Storage Conditions ........................................................... 3

1.4 Specifications ............................................................... 3

1.5 Additional Requirements ....................................................... 4

Chapter 2: Essential Practices ...................................................... 5

2.1 Storing and Preparing Samples and Reagents ...................................... 6

2.2 Priming and Loading the Chip................................................... 6

2.3 Running the Analysis.......................................................... 7

2.4 General Maintenance ......................................................... 7

2.5 Experion Video Tutorials ....................................................... 7

Chapter 3: Experion Pro260 Assay Procedure ......................................... 9

3.1 Set Up the Electrophoresis Station .............................................. 10

3.2 Equilibrate the Kit Reagents ................................................... 10

3.3 Filter the Gel and Prepare the Gel-Stain Solution ................................... 10

3.4 Prepare the Sample Buffers ................................................... 11

3.4.1 Reducing Conditions ..................................................... 11

3.4.2 Nonreducing Conditions .................................................. 11

3.5 Prepare the Samples and the Pro260 Ladder ...................................... 11

3.6 Prime the Chip ............................................................. 12

3.7 Load the Chip .............................................................. 13

3.8 Run the Pro260 Analysis...................................................... 14

3.9 Clean the Electrodes......................................................... 15

3.10 Evaluate the Run........................................................... 16

Chapter 4: Data Analysis ......................................................... 17

4.1 Viewing Data............................................................... 18

4.1.1 Managing Run Files and Project Folders in the Tree View.......................... 18

4.1.2 General Display Controls .................................................. 19

4.1.3 Electropherogram View.................................................... 19

4.1.4 Gel View ............................................................... 23

4.1.5 Results and Settings ..................................................... 23

4.2 Changing the Fluorescence Intensity Scale ........................................ 24

4.3 Using Results and Settings to View and Annotate Data .............................. 25

4.4 Comparing Data from Different Runs ............................................ 26

4.5 Saving, Exporting, and Printing Data............................................. 27

4.5.1 Saving Data Files ........................................................ 27

4.5.2 Exporting Data Files to Other Applications ..................................... 27

4.5.3 Printing Data Files ........................................................ 27

Chapter 5: Protein Quantitation Methods ............................................ 29

5.1 Protein Quantitation Methods .................................................. 30

5.2 Performing Percentage Determination............................................ 30

5.3 Performing Relative Quantitation ................................................ 31

5.4 Performing Absolute Quantitation ............................................... 32

5.4.1 Absolute Quantitation Using the Upper Marker as Standard ........................ 32

5.4.2 Absolute Quantitation Using a User-Defined Internal Standard ...................... 34

Chapter 6: Changing Analysis Settings and Parameters ................................ 35

6.1 Designating and Searching for Specific Proteins .................................... 36

6.2 Changing Protein Quantitation Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

6.3 Manually Setting a Marker..................................................... 36

6.4 Excluding a Peak from Analysis ................................................ 36

6.5 Changing Peak Finding Parameters ............................................. 37

6.6 Changing General Settings .................................................... 37

6.7 Baseline Modification ........................................................ 38

6.8 Turning Analysis Off ......................................................... 39

6.9 Manual Peak Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Chapter 7: Troubleshooting ....................................................... 41

7.1 Electrophoresis and Priming Stations ............................................ 42

7.2 Experion Pro260 Analysis ..................................................... 42

7.3 Contacting Technical Support.................................................. 46

Appendices .................................................................... 47

Appendix A: How the Experion System Works ........................................ 48

Appendix B: Deep Cleaning Procedure .............................................. 53

Appendix C: Chemical Compatibility ................................................ 54

Appendix D: Glossary............................................................ 58

Appendix E: Bibliography ......................................................... 60

Appendix F Ordering Information................................................... 61

1

Experion™ Pro260
Analysis Kit

1

Experion Automated Electrophoresis System

1.1 Product Description

The Experion Pro260 analysis kit is used for protein analysis with the Experion automated
electrophoresis system (Figure 1.1). The Experion system employs LabChip microfluidic technology
to automate protein and nucleic acid electrophoresis by integrating separation, detection, and data
analysis within a single platform. The Experion automated electrophoresis system uses smaller sample
and reagent quantities than standard analysis methods, and it accomplishes analysis in a single
30-minute, automated step.

The Experion Pro260 analysis kit supplies the microfluidic chips, reagents, and instructions required to
separate and analyze 10–260 kD proteins under denaturing conditions. The sensitivity of the Experion
Pro260 analysis kit is comparable to (though sometimes more sensitive than) colloidal Coomassie Blue
staining of SDS-PAGE gels. Each Experion Pro260 chip can analyze up to 10 samples.

For details about how the Experion Pro260 kit analyzes proteins, refer to Appendix A in this manual.

Register your Experion system in order to ensure you receive important updates on software,
tech notes, and manuals. Upon installation, a dialog will provide registration instructions.

4

1

2

5a

Fig. 1.1. The Experion system. The system includes the following components: 1) automated electrophoresis

station, 2) priming station, 3) vortex station used for nucleic acid analysis only, 4) system operation and data analysis
tools (software), and 5) analysis kits, which include the (a) chips and (b) reagents for protein (Pro260 kit), standard­sensitivity RNA (StdSens kit), high-sensitivity RNA (HighSens kit), and DNA (DNA 1K and 12K kits) analyses.

3

5b

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Experion Pro260 Analysis Kit

1.2 Kit Components

Table 1.1. Components of the Experion Pro260 analysis kit.

Item Description Volume (per Vial) 10-Chip Kit

Pro260 chip Microfluidic chips used for protein separation 10 chips

Cleaning chip Chip used for cleaning electrodes 1 chip

Pro260 gel Proprietary polymeric sieving matrix 520 µl 3 vials

Pro260 stain Proprietary fluorescent dye 45 µl 1 vial

Sample buffer Buffer for protein sample preparation; contains a 1.2 kD 400 µl 1 vial
lower marker and 260 kD upper marker for alignment of
samples to the Pro260 ladder

Pro260 ladder Protein standard containing 9 purified recombinant proteins 60 µl 1 vial
of 10–260 kD and optimized for automated electrophoresis
on the Experion system

Spin filters Used for filtering reagents during sample preparation 3 filters

1.3 Storage Conditions

Table 1.2. Storage conditions.

Item Storage Shelf Life

Experion Pro260 reagents 4ºC See expiration date on packaging

Experion Pro260 chips Ambient See expiration date on packaging

Gel-stain solution (GS, prepared) 4ºC 1 month from filtration (can be refiltered once)

Gel (G, prepared) 4ºC 1 month from filtration (can be refiltered once)

1.4 Specifications

Number of sample wells per chip 10

Sample volume required 4 µl

Total run time ~30 min per chip

Protein sizing range 10–260 kD

Limit of detection 2.5 ng/µl carbonic anhydrase (10 ng total) in 1x PBS; similar to
colloidal Coomassie Blue G-250 stain

Linear dynamic range 5–2,000 ng/µl (bovine serum albumin in 1x PBS)

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Experion Automated Electrophoresis System

1.5 Additional Requirements

Experion automated electrophoresis station

Experion priming station

Microcentrifuge (1,000–10,000 x g)

Heating block or water bath set at 95–100°C

Benchtop vortexer

Aluminum foil

Calibrated pipets and narrow-bore tips (for example, VWR #87001-688 or Rainin #L-10F)

Microcentrifuge tubes, 0.5 or 1.5 ml

Deionized water, 0.2 µm-filtered (ReadyPrep™ proteomics grade water, catalog #163-2091)

β-Mercaptoethanol (catalog #161-0710)

Benchtop centrifuge (optional, catalog #166-0612)

Extra spin filter for additional filtration steps (as needed, catalog #700-7254)

Experion electrode cleaner (catalog #700-7252)

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2

Essential Practices

5

Experion Automated Electrophoresis System

2.1 Storing and Preparing Samples and Reagents

Store all Experion™ Pro260 reagents at 4°C when not in use. Do not store reagents at room temperature
for >2 hr, as this will shorten their shelf life.

Before use, allow all kit reagents to equilibrate to room temperature (~15–20 min). Once thawed, gently
vortex all kit reagents before use. Before opening the tubes, quickly centrifuge them to collect solution
to the bottoms of tubes.

If the Pro260 gel has frozen, discard it.

Protect the stain, sample buffer, and gel-stain solution (GS) from light: store these solutions in a dark
place and keep them covered with foil when using them.

The Pro260 stain contains DMSO, which is hygroscopic. Cap tightly.

Use GS and filtered gel (G) for up to 1 month. After that, GS and G can be refiltered once again.

Prepare samples in either reducing or nonreducing sample buffer, but always prepare Pro260 ladder in
reducing sample buffer.

Do not use coated or treated pipet tips or microcentrifuge tubes (for example, siliconized polypropylene)
for preparation of kit reagents or samples. Use of treated tips or tubes may cause separation artifacts.

Use 0.2 μm-filtered or ReadyPrep™ proteomics grade water. Do not use autoclaved water for sample
or reagent preparation.

If possible, use sample protein concentrations (before preparation with the Pro260 sample buffer) that
are near the middle of the linear dynamic range.

2.2 Priming and Loading the Chip

To avoid contamination, wear gloves and handle chips by the edges. Never touch the glass portions of
the chip.

Load the chip on a benchtop or in the priming station. Never load a chip in the electrophoresis station.

Avoid sources of dust and other contaminants when preparing samples and loading the chip. Foreign
particles in reagents, samples, or the wells of the chip interfere with separation. Remove chips from their
packaging immediately before use.

It may be easier to load the chip on a white background. Tilt the chip to look for bubbles.

Use narrow-bore pipet tips for loading the chip (for example, VWR #87001-688 or Rainin #L-10F).

To avoid introducing air bubbles, do the following (for more help with chip loading, refer to the Experion
Training Video in the Experion software Help section under Contents and Index > Contents >
Appendices > Technical Videos):

n

Insert the pipet tip all the way to the bottom of the chip well when dispensing liquids

(this reduces the possibility of trapping air)

n

Hold the tip vertically, perpendicular to the chip surface. Holding the tip at an angle

may trap air bubbles at the bottom of the well

n

When expelling liquid, dispense slowly and only to the first stop on the pipet. Using the

second stop introduces air and bubbles into the liquid. Reverse pipetting is acceptable

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Experion Pro260 Analysis Kit

Dislodge bubbles at the bottom of a well with a clean pipet tip, or remove the solution and load it again.

Use a primed and loaded chip within 5 min of loading. When chips are not used within this time,
reagents may evaporate, leading to poor results or a chip performance error.

Fill all the chip wells when running an analysis. Use blank samples (prepared with water instead of
sample) or replicates if necessary. All 16 electrode pins must be in contact with liquid; otherwise, an IV
(current voltage) check failure error will occur.

2.3 Running the Analysis

Place the electrophoresis station on a stable surface, where it will not be subjected to vibrations or other
movement, and away from direct sunlight and all other potential sources of extreme heat.

Power on the electrophoresis station before launching Experion software.

The first time that the Experion electrophoresis station is used, confirm that communication has been
established between the software and electrophoresis station before preparing the reagents.

Do not open the lid of the electrophoresis station during a run. The run will abort if the lid is opened.

2.4 General Maintenance

For recommendations on general instrument maintenance, refer to the Experion system manual
(bulletin 10001312).

Clean the electrodes after each run (routine cleaning). Cleaning maintains the instrument in optimum
condition and prevents buildup and cross-contamination of reagents and samples.

Perform the deep cleaning procedure described in Appendix B to clean the electrodes:

n

Prior to first use of the Experion electrophoresis station

n

Whenever contamination is suspected or visible (for example, salt deposits or other

precipitates) on the electrodes

n

Whenever a chip has been left in the electrophoresis station for an extended period of time

(for example, overnight)

Never store the cleaning chip inside the electrophoresis station. Store the empty cleaning chip covered
to keep the wells clean. A new cleaning chip is included with every box of chips.

2.5 Experion Video Tutorials

For additional information, view the video tutorials available online at www.bio-rad.com:

North America: Home > Life Science Research > Support > Tutorials > Electrophoresis and Blotting >
Experion System Training

Other: Home > Life Science Research > Electrophoresis > Automated Electrophoresis >
Experion Training Videos

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Experion Automated Electrophoresis System

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Experion™ Pro260 Assay

3

Procedure

For an abbreviated version of this protocol, refer to the Quick Guide provided with the kit.

9

Experion Automated Electrophoresis System

3.1 Set Up the Electrophoresis Station

1. If needed, perform a deep cleaning of the electrodes (see Appendix B for instructions).

2. Power on the computer and then power on the Experion electrophoresis station by pushing the
green button in the center of the front panel. The steady green LED above the button indicates that
the unit is on.

3. Launch Experion software. If the instrument and computer are communicating properly:

n

A green dot and the last 4 digits of the instrument serial number appear in the lower right corner

of the software screen

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The electrophoresis station icon appears in the upper left corner

When there is no connection, these indicators are absent and a grayed-out instrument icon appears
in the upper left corner of the software screen.

3.2 Equilibrate the Kit Reagents

1. Set a heating block or water bath to 95–100°C. You will use this heating block to denature the
samples and Pro260 ladder later in the protocol.

2. Equilibrate the following kit reagents to room temperature for ~15–20 min:

n

Pro260 stain (blue cap)

n

Pro260 sample buffer (yellow cap)

n

Pro260 ladder (red cap)

n

2 tubes Pro260 gel (green cap)

3. Vortex the contents of each tube and briefly centrifuge the solutions to the bottoms of the tubes.
Make sure the Pro260 stain solution (blue cap) is thawed before proceeding.

If the gel-stain solution (GS) and filtered gel (G) were prepared previously, equilibrate them to room
temperature. Use the GS and G within 1 month of preparation. After 1 month, refilter them before
use. Keep both the G and GS at room temperature and covered until ready for use.

3.3 Filter the Gel and Prepare the Gel-Stain Solution

1. Prepare the GS by adding 20 µl Pro260 stain (blue cap) to a tube of Pro260 gel (green cap, 520 µl).
Vortex the GS for 10 sec at the highest setting and then spin it down briefly in a microcentrifuge.

2. Transfer the GS to a spin filter, and transfer the contents of the other Pro260 gel (green cap) into
another spin filter. Label and date the tubes.

3. Centrifuge both spin filters for 5 min at 10,000 × g. Inspect the tubes to ensure all of the gel passed
through the filters, and then discard the filters. Cover the GS with foil.

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Experion Pro260 Analysis Kit

3.4 Prepare the Sample Buffers

Separate protein samples under either reducing or nonreducing conditions, but always use reducing
conditions for separation of the Pro260 ladder. Reduced and nonreduced samples can be run on the
same chip. Prepare fresh sample buffer daily.

3.4.1 Reducing Conditions

For each chip, combine 1 µl β-mercaptoethanol and 30 µl sample buffer (yellow cap). Vortex and spin
down briefly. Protect the sample buffer from light.

At pH <7, reducing agents such as β-mercaptoethanol and DTT are less effective; use 5–7.5 mM
tributylphosphine (TBP) or tris(2-carboxyethyl)phosphine (TCEP) instead. Otherwise, neutralize the
buffer or use buffers of higher pH before preparing samples for Experion runs.

3.4.2 Nonreducing Conditions

Prepare two stocks of sample buffer: one reducing (for the Pro260 ladder) and one nonreducing
(for the protein samples).

1. For each chip, transfer 30 µl sample buffer (yellow cap) to 2 microcentrifuge tubes.

2. Add 1 µl β-mercaptoethanol to one tube (reducing sample buffer) and 1 µl deionized water to the
other (nonreducing sample buffer). This generates enough nonreducing sample buffer for
10 samples. Vortex the tubes and cover them with foil to protect them from light.

3.5 Prepare the Samples and the Pro260 Ladder

Each chip can analyze up to 10 protein samples. All wells of a chip must be filled for the
electrophoresis station to operate properly.

The total protein concentration, including any added user-defined internal standards, in all starting
samples should be toward the middle of the linear dynamic range of the assay, if possible.

1. Prepare the Pro260 ladder by combining 4 µl Pro260 ladder (red cap) and 2 µl sample buffer with
β-mercaptoethanol (reducing sample buffer) in a microcentrifuge tube.

2. Prepare the samples by combining 4 µl sample and 2 µl sample buffer (Section 3.4) in a
microcentrifuge tube.

3. Vortex all tubes briefly and spin down in a microcentrifuge for a few sec.

4. Heat the samples and Pro260 ladder at 95–100°C for 3–5 min. Spin down the tubes in a
microcentrifuge for a few sec.

5. Add 84 µl deionized water to each tube and vortex briefly to mix. Do not modify this step to adjust
sample concentration.

Both the diluted samples and the Pro260 ladder are stable for several hours when stored at room
temperature and protected from light.

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Experion Automated Electrophoresis System

3.6 Prime the Chip

Start the run within 5 min of priming and loading the chip. For help with chip loading, refer to the
Experion Training Video: Chip Loading, available in the Experion software Help section under
Contents and Index > Contents > Appendices > Technical Videos.

1. Pipet 12 µl GS into the top right well of the chip (highlighted and labeled GS, gel priming well)
(Figure 3.1). Insert the pipet tip vertically and to the bottom of the well when dispensing. Dispense
slowly to the first stop on the pipet, and do not expel air at the end of the pipetting step.

2. On the priming station, set the pressure setting to B and the time setting to 3, as specified by the
alphanumeric code on the chip (Figure 3.1).

Gel priming well

Priming code

Fig. 3.1. Experion Pro260 chip. The locations of the gel priming
well (GS, highlighted) and alphanumeric priming code are indicated.

3. Open the Experion priming station and place the chip on the chip platform, matching the arrow on
the chip with the alignment arrow on the chip platform. A post on the chip prevents insertion in the
wrong position. Do not force the chip into position.

4. Close the priming station by pressing down on the lid. The lid should snap closed.

5. Press Start. A “Priming” message appears on the screen of the priming station, and the timer counts
down. Priming requires approximately 60 sec. Do not open the priming station during countdown.

6. An audible signal and “Ready” message indicate that priming is complete. Open the priming station
and remove the chip. If the lid sticks, press down on it while pressing down on the release lever.

7. Turn the chip over and inspect the microchannels for bubbles or evidence of incomplete priming. If
the chip is primed properly, the microchannels will be difficult to see (it may be helpful to compare a
primed chip to a new, unused chip). If you detect a problem, such as a bubble or incomplete priming,
prime a new chip.

8. Place the chip on a clean surface for loading.

Bubbles forced into microchannels during priming take the shape of the microchannel and are
elongated, not round.

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Experion Pro260 Analysis Kit

3.7 Load the Chip

1. Using a pipet, remove and discard any remaining GS from the gel priming well. Pipet 12 µl GS into all
4 wells labeled GS (including the gel priming well, Figure 3.2).

2. Pipet 12 µl filtered gel (G) into the well labeled G (Figure 3.2).

3. Pipet 6 µl of each diluted sample into sample wells 1–10.

4. Pipet 6 µl diluted Pro260 ladder into the ladder well labeled L (Figure 3.2). Use the Pro260 ladder
within 8 hr of preparation. Every chip must have Pro260 ladder loaded into the ladder well labeled L.

Load 6 µl sample into wells 1–10

Load 6 µl ladder into well
labeled L

Fig. 3.2. Experion Pro26 0 chip. Wells for loading GS, G, samples, and ladder are indicated.

Load 12 µl GS into all 4 wells labeled GS

Load 12 µl G into well labeled G

5. Inspect all wells for bubbles by holding the chip above a light-colored background and looking
through the wells (Figure 3.3). Dislodge any bubbles at the bottom of a well with a clean pipet tip or
by removing and reloading the solution.

6. Place the loaded chip into the Experion electrophoresis station and start the run within 5 min.

Fig. 3.3. Bubble formation during loading of Experion Pro260 chips. Surface bubbles do not generally cause
problems during a run, but bubbles at the bot toms of wells must be removed. Lef t, bubbles trapped at the bottom of
wells. The GS and G wells and sample wells 1, 3, and 4– 6 contain no solution. Wells 8, 10, and L are filled properly and
have no bubbles, but large bubbles have formed at the bot toms of wells 7 and 9 (note the difference in the diameter of
the light-colored circles in wells 8 and 9). Right, bubbles have formed at the surface of the three GS wells on the right
side of the chip; the rest of the wells have no bubbles.

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Experion Automated Electrophoresis System

3.8 Run the Pro260 Analysis

1. Open the lid of the electrophoresis station by pulling the release latch. Place the primed and loaded
chip on the chip platform and close the lid.

2. In the Experion software toolbar, click New Run . In the New Run screen (Figure 3.4), from
the Assay pull-down list, select Protein > Protein 260.

3. Either select a project folder for the run from the Project pull-down list or create a new project folder
by entering a name in the Project field or by selecting File > Project > New. The project folder
appears in the project tree.

4. Enter a name for the run in the Run Prefix field and click Start Run .

Fig. 3.4. Details of the N ew Run screen. The green dot in the lower right corner indicates that communication
between the ele ctrophoresis station and Experion software has been established.

5. In the New Run dialog (Figure 3.5), select the number of samples to be analyzed. Though all wells
are filled, the Experion system stops the analysis when it reaches the number of samples entered.

Fig. 3.5. New Run dialog. The Experion system stops analysis when it
reaches the number of samples entered.

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Experion Pro260 Analysis Kit

6. Click Start. The green LED in the center of the front panel on the electrophoresis station blinks, and
the system performs a number of checks: it confirms that a chip has been inserted, that all wells
contain liquid, that electrical connections are made, etc. A calibration counter marks the progress of
these calibrations at the upper right of the screen.

Do not open the lid of the Experion electrophoresis station until the run is complete. The lid does

not lock. Opening the lid aborts the run.

An “IV Check Error” message indicates the system cannot make electrical contact in one of the
wells. This often means there is a bubble at the bottom of the well. Abort the run, and check the
chip for bubbles or empty wells. Refill the affected well(s), and start the run again.

7. During separation, the sample name is highlighted in the project tree and the electropherogram
trace, and virtual gel bands appear in real time:

n

The electropherogram of the sample being separated appears in the electropherogram

view

n

The lane corresponding to that sample is outlined in pink and has a dark background

To display the electropherogram of another sample, click on either the sample name in the project

tree or on a lane in the virtual gel.

8. When analysis is complete (after ~30 min), the instrument beeps and a window opens indicating the
end of the run. Select OK and remove the chip from the chip platform.

9. Clean the electrodes using deionized water within 30 min of each Pro260 chip run.

3.9 Clean the Electrodes

1. Fill a cleaning chip with 800 µl deionized water (0.2 µm-filtered). Gently tap the side of the cleaning
chip to remove any trapped bubbles from the wells.

2. Place the cleaning chip on the chip platform in the electrophoresis station, close the lid, and leave it
closed for 1 min.

Never store the cleaning chip inside the electrophoresis station. Store the empty cleaning chip
covered to keep the wells clean. A cleaning chip is included with each box of chips.

3. Open the lid, remove the cleaning chip, and allow the electrodes to dry for 1 min. Close the lid.

4. Replace the water in the cleaning chip after use to avoid contamination. For storage, remove the
water from the cleaning chip and store the chip in a clean location.

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Experion Automated Electrophoresis System

3.10 Evaluate the Run

When a run is complete, evaluate the run and the analysis of the data by Experion software.

1. Ensure all lanes (ladder and samples) are visible in the virtual gel. The markers (indicated by pink
triangles) should be visible in and aligned across all lanes. If the marker peaks are not properly
assigned, you may need to include or exclude peaks, or manually set the marker (see Section 6.3).

2. Evaluate the separation of the Pro260 ladder. To display the ladder electropherogram, click the
ladder well in the project tree, or click on the lane labeled L in the virtual gel. The electropherogram
should resemble the one shown in Figure 3.6 and should have the following features (if your ladder
does not have these features, see Chapter 7, Troubleshooting for more information):

n

Two marker peaks and a set of system peaks

n

Eight Pro260 ladder peaks between the system peaks and upper marker

n

Flat baseline

n

Marker peaks at least 20 fluorescence units above the baseline

Pro260 lad der pe aks

Lower marker

System p eaks

Upper m arker

Baseline

Fig. 3.6. Separation of the Pro260 ladder. Note
the flat baseline and well-resolved peaks. All
identified peaks are numbered, and the lower and
upper markers are indicated by green asterisks (*).
The 260 kD protein in the Pro260 ladder is labeled
as the upper marker in the Results table.

3. Examine the separation of at least one sample. Click on the sample name in the project tree or on
the lane in the virtual gel to view the electropherogram, which should have the following features:

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Two well-resolved marker peaks and a set of system peaks

n

Sample peaks located between the system peaks and upper marker

n

Flat baseline

If the upper and lower marker peaks are not properly assigned, you may need to include or exclude
peaks, or manually set the marker (see Section 6.3).

4. Evaluate the data analysis performed by Experion software (see Chapter 4, Data Analysis). If
necessary, change the analysis settings and parameters — including protein quantitation methods
— by following the instructions in Chapters 5 and 6.

16 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

4

This chapter outlines the basic steps and software features used to view and analyze data. For a
more detailed description of Experion™ software and its various functions, refer to the software Help
menu. For information on how to customize analysis parameters, refer to Chapter 6, Changing Analysis
Settings and Parameters.

Data Analysis

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