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Extracting proteins from cells


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#1 kellyly

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Posted 07 July 2009 - 05:30 AM

Hi all,

Im working with HUAECs, primary cells. Some of the proteins I'm interested in can be found in the cell and others are released into the medium during the growth of the cells in culture.
Could anyone advise me which methods are best to use to extract both types of proteins from these cells. The process needs to be compatible with the BCA assay and Western Blotting.
I have used Pierce M-Per reagent for lysing the proteins from the cells, I saved the medium from the flask but I don't know what method would be best or if even the M-Per one is good enough for the cells.

Many thanks.

#2 polyfractal

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Posted 25 July 2009 - 11:43 AM

We use a RIPA buffer for lysis, in combination with scraping. There are plenty of recipes for RIPA on the internet. It is basically a buffered detergent solution. I would use the following general protocol for your situation:

-Prepare RIPA buffer with fresh protease and phosphatase inhibitors
-Collect media and store for western blot
-Rinse cells with ice-cold PBS
-Add small quantity of RIPA buffer to cells
-Scrape, pipet into microfuge tube
-Let sit on ice for 30 minutes
-Centrifuge at some high RPM (we use 16k RPM, forget how many g's that is) for 30 minutes at 4 degrees
-Transfer supernatant to new microfuge tube.
-Add glycerol-based sample buffer, beta-mercaptoethanol, boil for 10 minutes

-Either store samples at -80 degrees C or proceed to gel electophoresis/western blot



Do not, however, that the protein content of the media may be very dilute and potentially too dilute to see by western blot. You may need to preciptate the protein in the media by some method and resuspend in a smaller quantity of buffer.




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