membrane protein aggregation - (Aug/10/2006 )
What are the usual methods to resolve the problem of membrane protein aggregation in SDS-PAGE?? This one is a combination of two protein subunits, one is 40kDa and the second one is 150kDa.
My problem is I am able to detect this protein in transfected cell (with the same antibody) but it is giving problem in native (primary) cells (sometimes there is a band at much higher molecular weight). Moreover, I never been able to get the band for regulatory protein (150kDa).
I have tried both boiling the sample and not boiling the sample before loading onto SDS-PAGE. Generally, I don't boil either in transfected or native cells.
Sample preparation is always done on ice with 1% triton-X-100.
I read that one should use peroxide free triton for membrane protein work. are my problem due to peroxide???
My Immunoprecipitation (overnight incubation with antibody) experiemtns with same protein are also not working.
thanks in advance for reply and explanation!!!!
Have you thought of native PAGE for the membrane proteins? I read, it should be used for difficult hydrophobic proteins. Or you could try to use another detergent, as SDS is very strong, perhaps Digitonin or Dodecylmaltosid? Just an idea...
may b ur protien is getting modified in the primary cells so u dont get the 150kD size band.