S35- met labelling of proteins: URGENT

Michael Benedik bchs1b at Elroy.UH.EDU
Thu Jul 30 23:30:17 EST 1992


In article <30JUL92.14472467 at wums.wustl.edu>, wetsel_r at wums.wustl.edu writes:
>That's an open question, some additional details would help.  However, we
>routinely label cultured cells using the 35S-Trans label from ICN.  We bring up
>the volume such that 100 ul equals 1mCi.  We plate our cells in either 24 or 6
>well plates at 3-4X10^5 or 1X10^6 cells per well, respectively.  Use met free
> medium with either 0.5 % BSA or 5% dialyzed calf serum.  Depending upon the
>cell type, we will add 0.250 - 0.500 uCi per ml of labeling media and incubate
>that on the cells anywhere from 2-24 hours, normally 4 hours at 37'C works very well for most continuous cell lines.  I should note, prior to adding the
>labeled media to the cells, we will wash the cells 2-3 times in HBSS.  This
>works very well in our hands for both adherent and non-adherent cells.  We oftenwill then use the corresponding supernatant and cellular lysate in
>immunoprecipitations - I'll assume that is what you're doing?  Post back
>if you need more help... best of luck...
>
>David

Is that a typo by chance, 0.250 uCi per ml. I would think that
250 uCi or (.250 mCi) would be more appropriate, although I am not an
expert at labelling tissue culture cells. We use 100 uCi per ml for
bacteria.
	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU



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