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anti-BRCA1 antibody specificity issues

BRCA1 western blot

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#1 Zam

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Posted 13 July 2012 - 06:35 AM

Hi,

I used three different antibodies for BRCA1 (Ab-1, Ab-4 and santacruz C-20). All of them gave a lot of non-specific binding and the blot looked almost like a ponceau staining. Among these three, Ab-4 seems to be less non-specific. I need to know does others also get lot of non-specific bands with any of these antibodies? I am having tough time determining the band corresponding to BRCA1. I used 2 different prestained markers, one starting from 250kDa and another from 170kDa. I get 3 - 5 bands inbetween 250 and 170kDa. I thinking of using blocking peptides. I even increased the washing steps with TBS-Tween. My control WB for other proteins seems to give clean bands.

Is it a known problem with the specificity of BRCA1 antibodies? or is it something that I am doing wrong?

I appreciate your suggestions.

Thanks

Zam

#2 bob1

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Posted 13 July 2012 - 07:22 PM

I think, from a colleague's comments, that it is common to cut either the gel or the membrane below the 100 kDa part to remove a large portion of the non-specific staining for BRCA. I don't know about which band is the specific one though.

#3 Zam

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Posted 16 July 2012 - 08:12 AM

Thanks Bob1. I do cut the membrane. yes, the problem is which band is specific around 170 to 250kDa range. I am planning to knockdown BRCA1 using siRNA and will check which band is altered with & without siRNA. But can safely conclude that no BRCA1 antibody is cleaner.




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