Bio-rad Experion Protein Analysis Kits User Manual

Experion™ Pro260 Analysis KitInstruction ManualCatalog #700-7101

2.1 Storing and Preparing Samples and ReagentsStore all Experion™ Pro260 reagents at 4°C when not in use. Do not store reagents at room temperature f

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 o

8 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.comExperion Automated Electrophoresis System

Experion™ Pro260 Assay Procedure3For an abbreviated version of this protocol, refer to the Quick Guide provided with the kit. 9

3.1 Set Up the Electrophoresis Station1. If needed, perform a deep cleaning of the electrodes (see Appendix B for instructions).2. Power on the com

3.4 Prepare the Sample BuffersSeparate protein samples under either reducing or nonreducing conditions, but always use reducing conditions for separa

3.6 Prime the Chip1. Pipet 12 µl GS into the top right well of the chip (highlighted and labeled GS, gel priming well) (Figure 3.1). Insert the pip

3.7 Load the Chip1. Using a pipet, remove and discard any remaining GS from the gel priming well. Pipet 12 µl GS into all 4 wells labeled GS (includ

14 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com3.8 Run the Pro260 Analysis1. Open the lid of the electrophoresis stat

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 15 Do not open the lid of the Experion electrophoresis station until

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

16 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com3. Examine the separation of at least one sample. Click on the sample

Data Analysis4This chapter outlines the basic steps and software features used to view and analyze data. For a more detailed description of Experion™

4.1 Viewing DataThe main window of the Experion software user interface contains menus and toolbars, a tree view, and three data views: the electroph

To open a run file, double-click on it. The run file opens, displaying the electropherogram and Results tab for the Pro260 ladder, and the virtual gel f

20 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.comSingle-Well ViewThis is the default view, which shows the electropherog

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 21Fig. 4.5. All-wells view. All electropherograms are shown in the elec

Peak LabelsExperion software automatically labels detected peaks with numbers for easy identification, and the upper and lower markers used for normali

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 234.1.4 Gel ViewExperion software converts electropherogram data into d

24 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com4.2 Changing the Fluorescence Intensity ScaleTo display more or less d

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 254.3 Using Results and Settings to View and Annotate DataThe followin

ContentsChapter 1: Experion™ Pro260 Analysis Kit ... 11.1 Product Description ...

26 Tech Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com4.4 Comparing Data from Different RunsExperion software allows you to overl

4.5 Saving, Exporting, and Printing Data4.5.1 Saving Data FilesData are automatically saved to a run file. Names for run files are given according to

28 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.comExperion Automated Electrophoresis System

Protein Quantitation Methods5 29

30 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com5.1 Protein Quantitation MethodsExperion™ software enables protein qua

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 315.3 Performing Relative QuantitationExperion software also automati

32 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com5.4 Performing Absolute QuantitationExperion software also allows quan

4. After evaluating the run as described in Section 3.10, select Analysis > Internal Std. and Std Protein Calibration Curve. 5. In the Internal S

34 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com5.4.2 Absolute Quantitation Using a User-Defined Internal StandardThis

Changing Analysis Settings and Parameters6Once an Experion™ analysis is complete and you have examined the quality of the run and of the data, you can

4.2 Changing the Fluorescence Intensity Scale ... 244.3 Using Results and Settings to View and Annotate Data ..

36 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com6.1 Designating and Searching for Specific Proteins1. Designate and s

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 376.5 Changing Peak Finding Parameters The parameters entered tell the

38 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com6.7 Baseline Modification To change the method used by Experion softwa

6.8 Turning Analysis OffTo turn analysis off, select Analysis > Turn Analysis Off. Analysis remains off for all runs until it is turned back on. T

40 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.comFig. 6.4. Manual integration mode indicates the beginning and end of pe

Troubleshooting7 41

7.1 Electrophoresis and Priming Stations Error Probable Cause Recommended ActionIV Check Failure(Experion™ software)One or more wells are not filled c

Error Probable Cause Recommended ActionNo peaks were detected in some samplesThere is not enough sample in the wells Ensure that pipets are calibrated

Error Probable Cause Recommended ActionReagents, specifically the Pro260 stain, were not brought to room temperature prior to use; if stain is not equi

Error Probable Cause Recommended ActionProtein quantitation is incorrectUpper and lower markers were not properly assigned by the softwareCheck that b

Experion™ Pro260 Analysis Kit1 1

Error Probable Cause Recommended ActionBaseline is fluctuating (for example into very negative and positive values)Air bubbles are interfering with ele

AppendicesA 47

Appendix A: How the Experion™ System Works The Experion automated electrophoresis system performs electrophoresis of samples within a microfluidic chip

How Experion Software Analyzes ProteinsData PresentationAs the photodiode detects fluorescent signals from the dye-LDS micelle-protein complexes, Exper

Once separation is complete, the software subtracts background noise, removes spikes, identifies and integrates peaks, and assigns their sizes and conc

Data Analysis: Normalization, Sizing, and QuantitationFollowing separation, Experion software analyses proteins using one or more of the following: n

of protein concentration, and the sample protein concentration is then determined from this calibration curve and reported in the the Calib. Conc. col

Appendix B: Deep Cleaning ProcedureThis manual procedure applies Experion™ electrode cleaner directly to the 16 platinum pins in the electrode manifol

Appendix C: Chemical CompatibilityFor best results, the starting protein sample (undiluted) should contain components at concentrations less than or e

Table C.2. Buffers with mild effects on Experion Pro260 separation or analysis. Buffer Concentration Effect(s)IEF anode buffer 1.5x: 10.5 mM phosph

2 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com1.1 Product DescriptionThe Experion Pro260 analysis kit is used for pro

Table C.5. Chemicals with mild effects on Experion Pro260 separation or analysis. Chemical Concentration Effect(s)Acetone 15% Quantitat

Table C.9. Detergents and reductants with severe effects on Experion Pro260 separation or analysis. Detergent/Reductant Concentration Effect(s)ASB-1

Appendix D: Glossary% Total Calculation performed by Experion™ software to show the percent of the total protein comprised by a particular peak; used

Normalization Alignment of protein peak migration times across different sample wells of the same chip; the lower and upper markers are used by Expe

Appendix E: BibliographyVisit us on the Web at www.bio-rad.com (search term “Experion”) for a list of technical literature and to download copies of a

Appendix F: Ordering InformationCatalog # Description700-7000 Experion™ System, 100–240 V, for protein analysis, includes electrophoresis station,

Life ScienceGroupBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 31.2 Kit ComponentsTable 1.1. Components of the Experion Pro260 analy

4 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com1.5 Additional Requirements Experion automated electrophoresis station

Essential Practices2 5