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Western Blot, Bax, Many bands

bax western blot

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3 replies to this topic

#1 smica602

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Posted 11 December 2012 - 03:59 PM

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.

Attached Thumbnails

  • WB2.jpg


#2 Curtis

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Posted 11 December 2012 - 05:39 PM

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.

#3 smica602

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Posted 12 December 2012 - 01:31 AM

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.


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/...1-antibody.html

Edited by smica602, 12 December 2012 - 01:33 AM.


#4 Curtis

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Posted 13 December 2012 - 06:22 PM

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.





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