nicotinic receptor western blot - a3 nAChR band at 100kDa instead of 55kDa (Sep/15/2010 )

I am using SCG lysates from rat pups to identify a3 nAChR by western blot. The primary antibody I am using is from Santa Cruz. I am resolving the proteins in 10% polyacrylamide gel and transferring the proteins onto a pVDF membrane. I am probing this membrane with primary antibody (0.5ug/ml). The band I am getting is at 100kDa instead of 55kDa. I know that sometimes the a3 nAChR protein can be identified at 60-62kDa region. I do not know what could be the reason. Suggestions regarding why this could be happening will be useful.

-mpaul-

Hola, I have seen the information about this antibody in the Santa Cruz web, and in the image of WB is the main band at about 50Kd and light signals of higher mol. weights as they were multimeric forms. Could it be possibly, in your case, of a partial denaturation showing a dimeric band. Does your loading buffer have reducer and it has been store in the fridge. Could be that it was oxidized. So, I would add more reducer and boil the sample. Good luck

-protolder-

mpaul on Wed Sep 15 22:07:31 2010 said:

In my experience antibodies for detecting nAChR subunits are notoriously crap. They are so crappy, in fact, that an article was actually written about it by Changeux and friends:

PMID: 17419810
J Neurochem. 2007 Jul;102(2):479-92.
Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.

My suggestion? Find another way to examine your question...

MM

-Mighty Mouse-