aspecific bands in cellculture protein, not in tissue - sds page (Jul/19/2005 )
I do a sds page on cellculture protein and as a positive control I have a tissue homogenate.
In the cellculture samples I get aspecific bands and no band for my protein of interest. In the tissue homogenate I get a nice clean clear band of the protein of interest.
How do I get rid of the background and see my protein of interest in the cell samples??
1. Did you use same buffer for your sample homoginization?
2. For your cellcultre sample, you can increase beta-mercaptoethanol and SDS concentration of your extraction buffer, if you just want to see the presence of your protein of interest.
I have similar experience as yours before, when I was using buffer A, I only got a false positive band; but when I use buffer A with 4% SDS and 1% beta-mercaptoethanol I can see my protien of interest clearly. Of course, the false positive still came out.
Thanks for your reply.
I do use the same sample buffer and this already contains 4% SDS and b mercaptaethanol.
I load about 10 ug of total protein. When I look at the blot just after blotting I can see the protein on the membrane for the cellculture sample and not for the tissue sample. Maybe this gives a hint for the solution to anyone??
I stripped the blot and put an actin antibody on. And hence, also this is shifted in the tissue sample. It seems like all the protein has run more quickly in the tissue sample. I get nice straight bands u can put your ruler to.
Strange these proteins in summer...