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Use Thermo Scientific SuperSignal Western Blot Enhancer to reduce background and enhance detection of low-abundance and weakly immunoreactive antigens. Insufficient washing. Increase the number of washes and/or the volume of buffer used. Add Tween 20 detergent to the wash buffer to a final concentration of 0.05%.
Example: Protein Degradation: Western blot analysis of extracts from NIH/3T3 cells, prepared in lysis buffer in the absence of protease inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37°C for the indicated time points, using β-Catenin (D10A8) XP ® Rabbit mAb #8480. In the ...
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The Western Blot Doctor is a self-help guide developed by Bio-Rad researchers that enables you to identify and troubleshoot western blotting problems. Comprehensive solutions and suggestions are provided to help solve your particular western blotting challenges. To start troubleshooting your western blotting problems, choose the type of problem ...
Since one issue that commonly arises during Western blotting is the presence of unusual or unexpected bands on the blot, our troubleshooting section begins with a table describing some common reasons and potential solutions for addressing and evaluating this type of problem . In most cases, the rest of the troubleshooting issues can be grouped ...
Increase the blotting time by 15 minute increments. Check the percentage of the gel used and switch to a higher percentage gel. Add 0.01–0.02% SDS to the transfer buffer to facilitate migration of the protein out of the gel. Decrease the amount of methanol in the transfer buffer.
This problem can be caused by antibodies binding to the blocking reagent in the blocking buffer. Change to another blocking reagent. Filter the blocking buffer. Wash excess detection reagent from the membrane before exposure. Overcome your Western blot difficulties with our troubleshooting advice, covering problems such as weak/no signal, non ...
The following troubleshooting guide is intended to explain causes and possible solutions for common problems observed in western blotting. Many of the challenges that require troubleshooting with traditional Western blots can be overcome with Simple Western™ which automates the traditional steps of Western blots inside of a benchtop instrument.
