I used muscle cells as sample, and the western blot band appear grey and blurry. I used non-fat milk for blocking, does this has any effect? This is my 10th time trying and the band still appear grey and blurry. I've done the protocol correctly, is there any other things I should consider doing or are there hacks for this problem? Thank you. Any advices will help a lot and appreciated.
Are you using streptavidin for detection? Nonfat milk contains biotin, so it should not be used as a blocking agent when probing with streptavidin.
If you can't get good results when blocking with milk, try using BSA or a commercial Western blot blocking agent.
High background may result if you are using an excessively high concentration of antibody, especially secondary antibody.
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