Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

Blocking buffer - (Mar/30/2010 )

What is everyone's favorite blocking buffer and why? I prefer 5% BSA in TTBS over 5% NFDM in TTBS as the 5% BSA gives a cleaner signal when I'm probing for phospho-proteins (which I usually am).

-Halfro22-

I use milk in TBS-T. Here we just use the home brand cheapest stuff, but when I was in England they were very particular that it had to be Marvel brand (apparently that is what works). I only use BSA for phospho-protiens, which is not too often.

-than4-

Generally 5% nonfat milk in TBS-T. If we're using any biotin/streptavidin systems, however, we use StartingBlock TBS-T because of the biotin present in nonfat milk.

-fishdoc-

5% nonfat milk in TBS-T here too...

-HomeBrew-

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.

-rkay447-

we usually use 2%bsa + 2%normal goat serum (ngs) in pbs to block and 2%ngs in pbst for our antibody solutions (our secondary antibodies are made in goat).

-mdfenko-

I believe this is an antibody issue, thus I will follow the instruction comes with the antibody. Some even states that you should use the milk not BSA or vise versa. For example, the EBioscience Trueblot anti-mouse IgG-HRP should not be used with BSA.

-goldfinger-

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?

-molecule-