question about lysis of cells - centrifuge or not--important? (Feb/15/2007 )

Hi,

I have been trying western blot to detect a protein, which upon expression, is secreted outside of the cells and bind to the extracellular domain of some transmembrane proteins. I have tried to analyze the total lysate and the concentrated medium, but failed to detect it in either case. I have been suggested that it may exist in the pellet formed after centrifugation of the cell lysate. (I have been using the RIPA buffer from Sigma, supplemented with protease inhibitor; I centrifuged the lysate at 8000Xg for 10min at 4C to pellet the cell debris as instructed in the product sheet.) I have also tried to resuspend the pellet and run western blot with that, but still failed to detect the protein of my interest. (BTW, I am pretty sure it is not the problem of the antibody.) I am thinking about to do western blot with the lysate directly without centrifugation. I am wondering what the harm that would do, since all the wb protocols that I read all recommend to centrifuge the lysate and get rid of the pellet of cell debris. Any advice would be really appreciated! Thanks in advance!

it could becasue the sample contains too low levels of the desired protein.

have you tried to make lysate without washing the cells. i am not sure but wondering if for some reason this protien which is bound to transmembrane protien might detach while washing.

Also try transient transfection and try to detect the protein, just so that you could verify that everything else is working.

good Luck !!!