Western Blot, Bax, Many bands - (Dec/11/2012 )

Hi,

Can anybody please give an idea as to why there are so many bax bands at many different sizes. Although supposed to be at 21 kDa, there are many more, some will be multimers with bcl-2, and other bax proteins, but there are just so many. Also one near the 17 kDa range?

Attached is a picture of a said blot. Red are bax, rabbit polyclonal, the other antibody here is a mouse monoclonal, so shouldn't cross react, (besides, when just looked at the green proteins, there is no overlap with the red).

Cheers,
Caine.

how do you block your membrane? I must say that it's normal to get nonspecific bands. and what is the name (clone) of your anti-Bax. My main area of work is Bax.

Curtis on Wed Dec 12 01:39:32 2012 said:

Cheers for your reply. I use a blocking buffer made by Li-cor (http://www.licor.com...ing_buffer.jsp#). We incubate our membranes at room temperature for 1 hour, and then with the primary antibodies, we add 10% BSA, and 10% NGS to the mix in PBS+tween20.

And, the bax antibody is from santa cruz: http://www.scbt.com/datasheet-6236-bax-delta-21-antibody.html

I have used 6A7, 2D2 and N20 clones. 6A7 wan't suitable for western blot, but the other two specially N20 were really great. I see nothing wrong with your method. I just think that it's normal to get nonspecific bands. and why do you expect to see multimers with Bcl2? Do you realize there are a lot of proteins that Bax binds to? Also, use TBS instead of PBS. For the incubation of primary antibody we just added ab+TBS.