When I detect phosphorylation and acetylation there is always non-specific band around 90kd which I suspect is caused by the 5% milk blocking buffer ( other antibody do not show similar bands) , I'v heard that BSA or gelatin might be better choice when detecting post-translational modificatoins ,does anyone have a suggestion ?
I would also recommend to not use milk powder for blocking. It's linked to show unspecific signals due to a relativly high content of phosphorylated proteins.
I worked with gelatin blocking buffer and it worked quiete good (small background though).
I don't have any experience with bsa blocking.
regards
5% BSA works very well for detecting phosphorylation. you might have to optimize the time. I too agree with Robert for not using milk.
Thank you both for answering this question ! I'm considering using BSA and gelatin for a test first to find a minimized background .
Although I might have some additional questions ---- what's the concentration of gelation used for blocking ? And will porcine gelatin serve our purpose as well as fish gelatin ？
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