Hi all
I have a problem with the PGC1 alpha/acetylated lysine western blot (with rat liver nuclear extracts).
Once I get the reading of the PGC1 alpha bands (with no problem, abcam ab54481), and after a stripping, I incubate my membranes with the appropriatte acetylated lysine antibody (cell signaling 9441).
The problem is that when I read the acetylated lysine, I get more than one band (a very "thick" one in the range of 150 kDa, but also other (weakers) around 100 and 75 KDa.
I would like to know which of these acetylated lysine bands is the one I have to use to correct the values obtained from the PGC1 alpha quantification.
Thanks for reading
I think you should use the thick band. Is there an expected MW for your protein? Can your protein exist as a dimer or something ? any possible disulphide bonds?
I would probably focus on the thick 150 kDa band. The other two are probably proteolytic (degredation) products. What is the m.wt of this protein? Do you include protease inhibitors when you make a nuclear extract?
Dear Keerthi and Haitham. Thanks for your answers. The mollecular weight of PGC1 alpha is about 105 KDa, and yes, I use protease inhibitors during the extraction.
It could mean that the acetylated protein binds another protein of about 45 KDa, which co-migrates as a complex on the gel with the 105 kDa protein!
Dear Mathew
What I am trying to measure is the acetylation status of PGC1 alpha, and for this I am using total PGC1 alpha and acetilated lysine antibodies.
Taking into account your explanations, I would have to repeat my WB´s and correct the PGC1 alpha readings with acetilated lysine readings that appears at the same molecular weight range.
Thank you very much
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