Membrane protein problem - Problem in getting signals (Nov/06/2008 )
I am working on a rare membrane protein called the tetraspanin. I bought this antibody from Protein Tech group (quite unknown). I boiled my samples (cell lines) with blue SDS loading buffer prior running to the gel.
I can't get the expected band. I read somewhere it is not very good to boil lysate if one is investigating membrane protein?
Any tips working with membrane protein?
Thanks alot for any input.
What do you mean "rare protein"? As in low level or specific cell-type expressing?
If low level expressed protein then maybe you did not load enough to get WB signal.
Is it a multi-TM proteins?
I boil my sample for membrane proteins detection too, no problem. Depend on protein I think. But if your protein is soluble in Triton, maybe try phase separation using TX-114 to concentrate the integral membrane protein for detection
I can't get the expected band. I read somewhere it is not very good to boil lysate if one is investigating membrane protein?
Any tips working with membrane protein?
Thanks alot for any input.
In some instances boiling membrane proteins can cause aggregation and the protein won't run correctly - might get caught at the top of the gel, etc. If your protein isn't expressed at very high levels you might want to try doing a membrane extraction which will essentially increase the amount that you are loading.
also, if boiling can cause aggregation of the protein then you can heat at 65C for 10-20 minutes.