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Thanks for your reply.
The samples that I used were whole cell lysates from cells 3 hours, 24 hours, and 48 hours post treated with 20 Gy of radiation, as well as cells without radiation as negative control.
So I think the signal should be strong.
By the way, what BSA solution do you suggest as blocking reagent? Do I need to use BSA solution for both primary and secondary antibodies?
Asked on Feb 21 2012
Thank you very much for your email.
We use 5% BSA (Sigma) in TBST for blocking and for diluting secondary and primaries. Milk in not recommended for phospho specific antibodies because milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Please be advised that BSA will only reduce the background i.e. membrane dirtiness, it will not effect the antibody specificity. So if the membrane shows high background then I would certainly recommend trying BSA.
The other important point is, whether the cells naturally express the protein or not. I am sorry the info of cell line wasn't included in your email so I can't be sure about it. I would however recommend using Hela lysates as positive control. Are you sing human cell line?
Actin or GAPDH can be used as loading control. Have you tried any of these?
I hope these suggestions will help to improve your results. If the results do not improve please do not hesitate to contact me.
Answered on Feb 21 2012