Hello, I am getting lots of unspecific bands when I do western blot for the phospho eEF2, (when I do it for the total eEF2, i get only one clear band) can someone suggest a specific technique for this antibody?
Hello Farah Issa
If you are detecting phosphoprotein by Western Blot, you shouldn't use nonfat dry milk as a blocking agent as it contains the phosphoprotein, casein which may react with the antibody resulting in non-specific binding and a high background.
You may use 5% BSA in TBST for blocking. Prepare antibody dilutions in 3% BSA in TBST.
Make sure you do not load excessive amount of lysate on the gel, antibody will bind non-specifically to proteins which is in excess resulting in multiple bands.
Try reducing the antibody concentration as higher antibody concentration may result in non-specific bands.
Lastly, sometimes poor quality of the antibody (contaminated or unpurified) could result in multiple bands. Try changing the primary antibody.
Hope this will help!
Good Luck!
No surprise. The anti-phospho-AB is detecting a phosphorylated motif, that apparently also is present on other proteins.
That's why you have to identify eEF2 with another antibody or isolate your target protein by immunoprecipitation.
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