Simple Western Size-based Assays

Simple Western Size-based Basics

A Simple Western by size is an automated Western—no gels, no transfer devices, no blots, no film and no manual analysis. Just load your samples in Wes, Peggy Sue, or Sally Sue and press start—it's a complete, walk-away solution for protein separation and detection.

  • Wes, Peggy Sue, and Sally Sue automate all steps automatically including sample loading, size-based protein separation, immunoprobing, washing, detection and data analysis.
  • Variability in the manual processes that used to impact reproducibility, quantitation, time to result and overall data reliability is eliminated.
  • Transition is simple—the same antibodies used for Western blotting protocols can be used with Simple Western assays.
  • Up to 96 samples can be processed at once, and assays take just 19 hours to complete with sizing and quantitation results.
  • Separate and detect proteins as large as 440 kDa in as little as 3 hours.

How Does it Work?

With the addition of the Total Protein Assay, there are now two ways to run Simple Western size-based assays—immunoassay or total protein. Just load your samples and off to the races you go!

Simple Western immunoassays take place in a capillary. Samples and reagents are loaded into an assay plate and placed in Wes, Peggy Sue, or Sally Sue. Proteins are loaded into the capillary automatically and separated by size as they migrate through a stacking and separation matrix. The separated proteins are then immobilized to the capillary wall via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and immunoprobed using an HRP-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is detected and quantitated.

Immunoassay

Total Protein Assay

Interested in looking at all the proteins in your lysate? Just leave out the antibody and let Wes, Peggy Sue or Sally Sue do the work for you. Target proteins separated by size are labeled with a biotin reagent and is detected by chemiluminescence using Streptavidin-HRP. At the end of the run, you’ve got relative quantitation for your protein of interest in total protein in the sample.

What Does the Data Look Like? Simple Western size-based assay data is processed automatically in Compass software. Sample data is displayed by lane in a virtual-blot like image similar to traditional results with one big exception—not only do you get more information, you get it as soon as the assay is complete. Quantitative results such as molecular weight, signal intensity (area), % area, and signal-to-noise for each immunodetected protein are presented in the results table automatically.

3 pro gel

For those more familiar with capillary electrophoresis, a more traditional electropherogram view is also available.

electropherogram

 

Looking at big proteins? Try this on for size. Simple Western now has a wider molecular weight range capability, so you can detect proteins as large as 440 kDa.

Detection of 3 high molecular weight targets.
HECTH9, Talin and UBR5 were detected in K562 lysate using the 66-440 kDa Master Kit.

 

Reproducibility and Quantitation

Simple Western assays are fully automated by Wes, Peggy Sue, or Sally Sue and easily standardized, making results much more reproducible. There is no blotting step, so inconsistencies caused by protein transfer are eliminated, vastly improving quantitation.

Phosphoinositide 3-kinase (PI3K) expression in an MCF-7 lysate. Ten replicates (n=10) of MCF7 lysate at 1 µg/µL were analyzed using a PI3K-specific primary antibody. The predicted molecular weight of PI3K is 110 kDa. Simple Western assay data reported an average molecular weight of 107 kDa with a 0.5% CV for sizing and an 8.7% CV for signal intensity (area).

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PI3K table

Dynamic Range

Simple Western assays have a linear dynamic range of approximately 3 orders of magnitude, allowing detection over a wider concentration range with more accurate protein quantitation.

dynamic range

Dynamic range of detection for GSK-3 alpha. Lysate from K562 cells lysed in Bicine/CHAPs buffer was serially diluted and analyzed using a GSK-3 alpha antibody. The calculated dynamic range was 3.3 logs with an R² value of 0.9995.