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How can I get optimal lysis of COS1 cells


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#1 Trying-2-Graduate

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Posted 14 July 2009 - 10:33 AM

Here is what I am trying to do: I am incorporating an azide-moiety on to proteins in situ. I would then like to lyse the cells, separate the cytosolic proteins from the membrane-bound, re-suspend the membrane bound, and then analyze the two samples (cytosolic and membrane) for azide-modified proteins. I will use click chemistry to enrich for the labeled proteins, then do LC-MS/MS on the digests to identify them.

Now, here is my problem: I am not getting "good" cell lysis. I was told that for mammalian cells, a good estimate of 100ug protein can be obtained from 1x10^6 cells. I only obtained about 1/10 of that when I used the following procedure for COS1 cells:

Suspend cells from 1x 100mm plate (~ 5x10^6 cells) in 250 uL PBS + protease inhibitors and put on ice
Sonicated with a Fisher Scientific* Model 100 Sonic Dismembrator set at ~ 20% power for 10 sec pulse with 30 sec break between pulses. I did a total of 3 pulses.

Am I not pulsing long enough? I could go a little higher in power, but foaming started to occur when I did.

Someone told me to add SDS (like at 1%) to the PBS, but won't that cause the membrane associated proteins to not be separated from the cytosolic?

I am at my wits end here, so any advice on how to get the best lysis possible that would allow be to do what I would like would be wonderful.

THANKS! smile.gif




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