Detach cells and stop stimulation simultaneously- How?
Posted 29 February 2012 - 12:12 AM
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?
Posted 29 February 2012 - 12:55 PM
Posted 01 March 2012 - 02:35 AM
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?
Posted 01 March 2012 - 12:52 PM
Posted 12 March 2012 - 01:42 AM
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.