Hello,
Our lab mixes primary antibodies with BSA and Sodium Azide and I wonder what purpose BSA serves. I understand that BSA is used for blocking buffers. But the membrane is already blocked with skim milk. Is BSA just for additional blocking? Is BSA essential?
Thanks
Dear Andrew Kim
BSA is used extensively as a carrier protein to dilute antibodies and as a general protein-blocking agent in immunoassays and immunodetection protocols. Putting it in a BSA solution allows the antibody to be reused if the blot does not give a good result. The concentration of the antibody depends on the instruction of the manufacturer. The antibody can be diluted in a wash buffer. But you can use only TBS-T for antibody dilutant. Then, I recommend not re-use.
Article Western Blot: Technique, Theory and Trouble Shooting
Kind regards
AB Bayazid
Very dilute proteins, including antibodies, can be lost due to adsorption to the surface of the container, causing inconsistent results, so it is a good idea to include some other substance to block surface binding. BSA is often used for this purpose because it is a relatively inexpensive protein. The blocking buffer containing milk (casein) would probably work just as well.
A.B Bayazid so you're saying essentially BSA acts like a preservative for the antibody?
Adam B Shapiro I just find the BSA as antibody diluent redundant if there is going to be a blocking step w/ skim milk anyways. Just a general question though, can the BSA used to dilute antibodies interrupt western blotting? I've heard from one of the Ph.Ds in the lab that BSA can sometimes hinder the protein band that we want to see.
Andrew Kim
I didn't say this. BSA helps to stabilize antibody proteins and disperse them.
I have never heard of BSA in the diluent causing interference in Western blotting.
It would be a problem if it were present at a high level in the sample that is on the blot, such as in fetal bovine serum-containing growth medium, because it is so hugely abundant.
A.B Bayazid ahh gotcha! thanks for the answer! Helped shed light on my knowledge indeed!
Adam B Shapiro thank you always for the insightful answers!
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