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SIMPLE WESTERN CHARGE-BASED Separation
The Simple Western charge assay is an automated assay that doesn’t require the use of gels, transfer devices, blots, film or manual analysis where proteins are separated by their isoelectic point (pI) rather than their molecular weight. Charge-based assays (using isoelectric focusing, or IEF) can provide enhanced sensitivity and exquisite resolution for nearly all variant forms of a protein. Because proteins are separated by their pIs, you'll be able to see extremely small isoelectric point differences, to resolve post translational modifications such as phosphorylation and glycosylation that changes a proteins pI. It's a new way of looking at discrete changes in very small samples that you might just be surprised by!
how does charge-based separation work on Simple Western?
Simple Western charge-based assays take place in a capillary. Just load your samples and off to the races you go! Samples prepared in ampholytes and required reagents are loaded into an assay plate and placed in Peggy Sue or NanoPro 1000. The prepared sample is automatically loaded into the capillary and separated by charge according to their pI before the resolve proteins are immobilized to the capillary wall via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and detected using an HRP-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is detected and quantitated.
WHAT DOES THE CHARGE-BASED ASSAY DATA LOOK LIKE?
Simple Western charge-based assay data is processed automatically in Compass for SW software. Sample data is displayed as an electropherogram where it's easier to see minute changes. Quantitative results such as pI value, signal intensity (area), % area, and signal-to-noise for each immunodetected protein are presented in the results table automatically.
From Your Peers
“The Simple Western Charge assay, a capillary isoelectric focusing immunoassay, exceeded the reliability of 2D Western blots for resolving recombinant PKG-Iα and PKG-Iβ and uses 100,000X less sample quantity, making it well-suited for clinical disease proteomics because protein isoforms and post-translational modifications can be detected in precious tissue biopsies.” - Mary G. Johlfs, M.S., Director of Research Operations/Scientist, Roseman University of Health Sciences
Learn how researchers at Manchester University have developed. assays encompassing clinical samples from Chronic Myeloid Leukaemia (CML-SC), Chronic Lymphocytic Leukaemia (CLL), Non-Small Cell Lung Cancer (NSCLC; tissue & plasma) and Endometrial Cancer using ProteinSimple’s Simple Western charge assay. This and other work with Protein Simple defines the utility of the Simple Western charge assay in detailed mapping of signaling cascades in model systems and cellular organelles.
Additional Simple Western Resources
Publication Spotlight - Simple Western Charge Assays are Enabling Cutting Edge Biomedical Research