Detach cells and stop stimulation simultaneously- How? - (Feb/29/2012 )
My purpose is to do a FACS analysis of a receptor tyrosine kinase on the cell surface of an adherent cell line. I want to do the analysis when the receptor has been ligand stimulated for 5 min. What should I do to stop the reaction at 5 min?
Should I add cold PBS and put the cells on ice and then scrape them of the plate?
That's about the only way to do it - note that scraping will extensively damage your cells, so it may not be the best option. You may be able to fix the cells and then detach with trypsin.
Thanks for the answer, bob1!
Since my interest is to quantify the receptor expression on cell surface, is it possible to do this through western blot.
For example, I could stimulate the cells for 5min and then stop the the reaction with cold PBS and putting the plate on ice. While incubating on ice, I would add primary antibodies against the N-terminus of the receptor and after X minutes, wash off the antibodies and lyse the cells.
Transfer the cell lysate to eppendorf tube, add protein beads to immuniprecipitate antibodies bound to my target and subsequently do a western blot of the IP product.
How does this sound?
You might be better off stopping the stimulation then performing a membrane fractionation. I think that the procedure you describe above would probably not work particularly well as the antibody/protein interaction would probably be disrupted by the lysis process, unless you are very careful with the lysis conditions.
Just for the sake of completing this thread, I’ve decided to do a cell surface receptor biotinylation assay. Basically, incubating cells with biotin (in cold PBS) which binds to all available primary amines on the cell surface. After 1h of incubation, un-bound biotin will be inactivated by incubating the cells in 50mM Tris pH 8 for 5 min. After that, I will lyse the cells and precipitate surface proteins with streptavidin agarose. Finally, the cells surface receptor will be visualized through western blotting.