![]() ![]() Overall quantitation is vastly improved as blot transfer is not required, thus eliminating any inconsistencies in protein transfer. Because the Simple Western assay is fully automated, results are more reproducible than those generated via Western blot. Reproducibility of results from a traditional Western blot is a common challenge for researchers due to lack of standardized procedures and the multiple handling steps that introduce experimental variability. More quantitative and reproducible results ![]() The software reports molecular weight, area, percent area and signal to noise for each protein detected. Simultaneous analysis of up to 12 samples can be performed in a single experiment, and results are available in 3–5 hours. Molecular weight and signal for immunodetected proteins are automatically reported. Target proteins are then identified with a primary antibody and subsequent immunodetection using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate. The separated proteins are immobilized to the capillary wall via a proprietary, photoactivated capture chemistry. Proteins are separated in capillaries as they migrate through a stacking and separation matrix. Simple Western assay buffers, nano-volume capillaries and the prepared assay plate are placed in Simon, which carries out all assay steps automatically. The prepared samples, primary and secondary antibodies and chemiluminescent substrate are dispensed in microliter volumes into designated wells in a low-volume 384-well assay plate. Samples are then mixed with Simple Western Sample Buffer and standards to a final concentration of 1 μg/μL, reduced and denatured. Samples are prepared following conventional procedures 3. Manual factors that can negatively impact reproducibility, quantitation, time to result and overall reliability of the generated data are eliminated. The Simple Western is a reinvention of the entire Western blot, automating all steps from protein loading and separation, immunoprobing, washing, detection and quantitative analysis of data, finally giving researchers a complete, walk-away solution. Improvements have been made to reagents and individual steps within the Western blotting process over the years, but none has fully overcome the challenges and bottlenecks still experienced by researchers today. Although Western blotting is a proven technique, it is plagued by poor reproducibility, lack of accurate quantitation, extensive time to result and reliability issues. To avoid under- or overloading samples, determine the protein concentration of each sample prior to electrophoresis with a compatible protein assay.Western blotting is the most widely used and accepted methodology for protein detection and was first reported in the literature over 30 years ago 1, 2.However, if one cannot change the gel electrophoresis chemistry system, one may need to perform sample clean-up to render the sample compatible with the given system. Selecting a gel electrophoresis chemistry that is compatible with the buffer one’s sample is prepared in, is the simplest route. Some buffer components may interfere with the chosen gel electrophoresis chemistry system (e.g., Tris-glycine, Bis-Tris) and cause a variety of artifacts when running the gel.To prevent these negative effects, protease and phosphatase inhibitors should be added to the lysis reagents. Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and lead to degraded proteins.To minimize sample variability, keep sample preparation workflows simple, and use reagents optimized for the specific sample type and target proteins. ![]()
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