We have been having problems with high background in our western blots for phospho-proteins even though we are using BSA. We found that certian lots of BSA give high background. What are your inputs? we use TBST.
Milk contains a number of different proteins one of which is the phosphoprotein casein. This phosphoprotein can lead to higher background due to non-specific recognition of the phospho motifs. BSA is thought to work better for phospho antibodies as albumin is a secreted protein and tends not to be phosphorylated. However, despite this general rule of thumb, blocking with BSA does not always work better than milk for phospho antibodies. Generally when using a phospho antibody I'd start with BSA blocking and if that doesn't work I'd switch to milk (for example, pS6K1 T389 clone 108D2 from Cell Signal is cleaner if the membrane is blocked in milk, rinsed a few times then probed in BSA). If neither of those work then there are commercially available non-protein blocking agents available.You can also make your own non-protein blocking agent using polyvinylpyrrolidone (http://www.ncbi.nlm.nih.gov/pubmed/8095775).
from Christopher M. Bartley
Western blotting - using BSA or milk? And TBS-T or PBS-T? - ResearchGate. Available from: https://www.researchgate.net/post/Western_blotting-using_BSA_or_milk_And_TBS-T_or_PBS-T11
Either you can reduce the % concentration of your blocking agent and or you can switch using non-protein blocking agents. On my experience 3-5% BSA works better for phospho-protien detection.
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