Problems with histones in SDS-PAGE (View forum version)



SESC

Posted 26 September 2014 - 07:48 AM

Hello,

I'm asking for help because I have a problem I have been trying unsuccessfully to resolve. I extracted chromatin from cell who express a recombinant H2A histone and then extracted total histones from this chromatin and then I made a SDS-PAGE. I used a comercial sample of histones (Sigma) as control. I know that histones are not well separated in SDS-PAGE and that TAU gel is the most recomended for this purpose but I just need to identify that my recombinant protein is inside the chromatin and I expect do this by Western blotting, it's a control. My problem is that when I run the electrophoresis the samples seems to not enter correctly in the separating gel, and when I see the proteins there is only one difuse band. In several protocols I read that histones could be resuspended in water and separated in a SDS-PAGE but I can't do it!!! I changed loading buffer, solutions for gel preparation, I tried diluting histones in PBS, and the result is the same.

 

I appreciate you could give me suggestions or tips.

 

Thanks in advance!!!


mdfenko

Posted 06 October 2014 - 04:44 AM

can you post a picture and give us your procedure (especially preparation of sample for electrophoresis)?