S35- met labelling of proteins: URGENT
David I Resnick
dresnick at athena.mit.edu
Thu Jul 30 19:00:28 EST 1992
In article <13702 at gazette.bcm.tmc.edu> ak990140 at SPECIALK.IAIMS.BCM.TMC.EDU (Arthur Kania) writes:
> I'm trying to label proteins secreted by cultured cells. I would
> like to know how much labelled amino acid I should add in order to
> label adequate amounts. The medium is already met-deficient.
It depends on the cell line/how heavily your protein expressed/how
many methionines your protein has. We do the following for proteins
secreted from CHO cells. Note that if you want to label for very long
periods (overnight), you may have to add a bit of cold methionine as
well, or your cells will actually consume all of your 35S-met, and
stop producing protein.
1) Day 0:
Set cells @1.5x10^5 per well in 6 well dishes
2) Day 2:
Wash cells 2xPBS (to remove free methionine!)
Add labelling media
80 uCi/ml 35S-Met or 35S-Express mix
5% DIALYZED FBS (dialyze vs 150 mM NaCl, sterile filter)
You can leave this out. The cells are perfectly happy
without serum for reasonable periods of time
Incubate 5 hr, harvest media, spin at 1500xg to remove floating cells.
Notes and thoughts
The quantity of 35Smet that you use and the length of time that
you label for obviously will be determined by the level of expression
of your protein. We have been using stuff from New England Nuclear
called 35Express (or something like that). It is a hydrolysate of
proteins from bacteria which have been fed 35SO4. It is relatively
cheap - $99 for 5 mCi.
> Thanks for Your help.
David Resnick dresnick at athena.mit.edu
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