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Detach cells and stop stimulation simultaneously- How?


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4 replies to this topic

#1 Gangwolf

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Posted 29 February 2012 - 12:12 AM

Hi all!
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?

#2 bob1

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Posted 29 February 2012 - 12:55 PM

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.

#3 Gangwolf

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Posted 01 March 2012 - 02:35 AM

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?

#4 bob1

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Posted 01 March 2012 - 12:52 PM

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.

#5 Gangwolf

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Posted 12 March 2012 - 01:42 AM

Thanks bob1!
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.




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