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What should I do if there are many bands in Western Blot?

Mengyuan Singh Oct 18, 2022

Recently, when I was doing Western Blot, I found that although my background is relatively clean, there are always a lot of non-specific bands here, and sometimes these bands will gather near the bands of my destination, which will affect the display of results. What's going on?

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gabi Karash Answered Oct 19, 2022

Harvard University

1. May be there is protein degradation, you can use fresh samples to experiment, and add protein inhibitor to the protein extraction solution.

2. The antibodies concentration is too high, you can appropriately reduce their concentration (especially primary antibody).

3. Primary antibody has a poor specificity, it is recommended to replace the primary antibody.

4. There is a non-specific binding of the secondary antibody, and the control group can be set to exclude interference.

5. Protein may have dimers or multimers, you can boil for 10 minutes before loading to make the protein fully denatured.

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