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ProteinSimple : Western Blots ReInvented |
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Simple Western Assays
- Simple Western Basics
- Reproducibility and Quantitation
- Dynamic Range
A Simple Western is an automated Western—no gels, no transfer devices, no blots, no film and no manual analysis. Just load your samples in Simon and press start—it's a complete, walk-away solution for protein detection.
- Simon automates 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 are eliminated.
- Transition is simple—the same antibodies used for Western blotting protocols can be used with Simple Western assays.
- Up to 12 samples can be processed at once, and assays take just 3-5 hours complete with sizing and quantitation results.
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How Does it Work? Simple Western assays take place in a capillary. Samples and reagents are loaded into an assay plate and placed in Simon. 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.
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What Does the Data Look Like? Simple Western 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 is presented in the results table automatically.
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Simple Western assays are fully automated by Simon and easily standardized, making results much more reproducible. There is no blotting step, so inconsistencies caused by protein transfer are eliminated, vastly improving quantitation.
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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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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 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.
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Automated Western Blots
