How to harvest cells for phosphoproteomics

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chengh1
Glycine Member
Glycine Member
Posts: 5
Joined: Tue Jun 24, 2014 11:12 pm

How to harvest cells for phosphoproteomics

Postby chengh1 » Sun Jul 27, 2014 7:20 pm

Hi all:

I am confused of how to harvest cells for phosphopeptides enrichment, because we are treating cells for short time, only 5min, but it is always recognized that phosphorylation happens quite fast and also we are trying to characterize the early signalling. So I am wondering should we treat cells with our drugs and then snap freeze them with dry ice or should wash with ice-cold PBS and then harvest them directly? Welcome all the suggestions and experience.

Thanks a lot.
Cheng

ranio
Angiotensin Member
Angiotensin Member
Posts: 31
Joined: Fri May 04, 2012 1:06 am

Postby ranio » Wed Aug 06, 2014 9:25 pm

Hello Cheng,

Here is a similar work from Heck lab, they treated T-cells with PGE2.
Before PGE2 stimulation, cells were centrifuged for 1 min at 1500g, growth medium was removed and the cells were resuspended at a final concentration of 1‐2 × 106 cells/ml in RMPI. Next, cells were supplemented with 0 (control) or 10 μM PGE2 and incubated for 5, 10, 20, 30 or 60 min. After treatment Jurkat cells were washed twice with PBS and harvested, ell lysis was performed on ice.


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