In my western blot analysis of both total cell lysate and immunoprecipitated samples, I saw 2 bands. The expected band is around 15kDa but I see another additional band around 25kDa in every western blot that I performed. The protein that I am working on is flag tagged. I thought it might be a post-translational modification, but since the difference is 10kDa, it is unlikely. Additionally, the protein is an endosomal protein. If you have any idea about the reason please help me.
Thank you.
Hi, thank you for your response. The problem is my expected length is around 15kDa, but the unexpected length that I see is at 25kDa. As you said if the protein has shorter transcript variant, my expectations would be vica versa. I thought there might be a problem in the lysis solution. It might be very unlikely but, since my protein is an endosomal membrane protein, is it possible that some of the product is still attached to lipid content so that it can not migrate through the gel as fast as the unbound one, since my lysis buffer is not very strong?
If u lyze the cell pellets directly in 2x gel loading dye, may be you can exclude all sort of denaturing/membrane attachment issues.
Some additional questions which could help in trouble shooting would be: Which cells are u expressing? DO you blot using flag antibodies or with protein specific antibodies? D you really have very similar bands in IP as well as total cell lysates? Is it N or C terminal flag? Do you have stop/start codon cloned?
Have you looked at the possibility that you have dimers present? Stronger denaturation or reducing conditions might help avoid this possibility.
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