rkay447, on Mar 31 2010, 10:03 AM, said:
I usually use 5% milk/TBST for non-phospho antibodies and 5%BSA/TBST for phospho-antibodies. I just wanted to add that sometimes it is important to use PBST however!! I was having issues with an antibody and on the advise of a labmate, I ran two equal gels of lysate. One membrane was treated completely in 5% milk/PBST (block and blotting), the other in 5% milk/TBST. Same antibodies, same dilutions. I got NO signal with the TBST but huge and clean signal with the PBST. I did a control actin blot (in TBST) on both just to show that the loading was the same and that the membrane that gave me no signal with the first antibody in TBST could be probed with TBST...it was an antibody preference. I have no idea why there was such a huge difference but now I know that if I want to probe with this antibody (it's against Cdc45 by the way) I must use PBST. If you are having problems it may be worth it to try a different buffer.
what is the starting concentration or % of PBST that should be used to make the blocking buffer?