I am trying to perform a western blot to detect the S/T Kinase ATM in it's dimerized state. The 350 kDa homo-dimer is linked by 2 or more disulfide bridges, and I've detected the 175 kDa monomer in fully denaturing and reducing conditions. I want to probe for the dimer from fresh lysate. I know I can remove 2-mercaptoethanol from my loading buffer which is reducing. Is SDS itself reducing in any way? What about boiling, will this break the disulfide bonds? The company I bought my ATM antibodies from said that they obtained their flawless 350 kDa signal in fully denaturing and reducing conditions, but I'm skeptical, especially since I saw primarily the monomer in those conditions (but from repeatedly frozen and thawed lysate). Any other tips for blotting for very large proteins/dimers are also welcome, thanks!
The disulfide bonds will not be reduced by SDS or by heating in the absence of any reducing agent. Just prepare or buy some sample buffer without 2-mercaptoethanol.
350 kDa is very large for SDS-PAGE. You will probably want to run a low-percentage Tris-acetate gel.
Thank you! I am using a 7% Tris-Glycine gel and performing a long wet transfer to PVDF at 4C. What is the advantage of the Tris-Acetate gel?
Tris-Acetate gels are recommended for separation of very large proteins.
Dear
I suppose that 4 conditions are required for non reducing western blots :
You need: Net charge of protein of interest, Thermostability, Protein size.
and Transfer conditions.
Thank you
Marius
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