I am trying to detect proteins using Western Blot Technique. Due to the size difference, smaller protein of 36kDa was not seen on the blot read-out. Would gradient gel be a better idea to detect such proteins?
I think a 4-12% or 4-20% gel should work for you
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5871D.pdf
I think a 4-12% or 4-20% gel should work for you
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5871D.pdf
Gradients should work, although you should be just well with 10%. You only need to optimize the running time, so the 34 kD protein is still in the gel. In my experience, you can catch ~30 kD and ~210 kD on 10% pretty easily, given your antibodies work well and you don't need much separation on the bigger protein.
I've used Bio-Rad's 4-12% gradient gels to effectively separate out bands in the 20kDA through 280 kDA range.
The only thing I will note is that if you're then trying to transfer both of them for quantitative analysis, you'll need to make sure you have the right protocol which will ensure complete transfer from your gel to the membrane without leaving any protein behind on the gel or allowing substantial protein (particularly the lower MW band) to transfer through the blot.
i definitely support Nikolais statement, that the SDS PAGE isn't the problem and you should check your blotting protocol. Maybe you can also cut the gel and blot both bands with optimized conditions.
Thank you all for the answers, I am trying 4-20 gradient gel today and I will see how that works !
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