Metabolic labelling

Bob Steinberg rsteinbe at etowah.uokhsc.edu
Thu Mar 13 12:58:14 EST 1997


L.Kempster wrote:
> 
> Can anyone tell me how 35S metabollic labelling time is determined?  I have
> read several variations from 15 minutes to several hours.  I myself have
> tried 15 minutes, 1 hour and 20 hours.
> 
> Also, when pre-incubating with methionine-free medium and subsequent
> incubation with 35S-Met medium should fetal calf serum be included or not
> i.e. does FCS contain methionine or cysteine?
> 
> Thanks in advance,
> 
> Lee Kempster
> Imperial College
> 
Labeling times depend on what you are trying to accomplish: labeling
times much shorter than the protein's half-life give labeling
proportional to the protein's rate of synthesis; longer labeling times
give labeling that approaches proportionality to the steady-state level
of the protein.

Serum contains methionine, and methionine is necessary for protein
synthesis. We have found that 10% serum in methionine-free medium
provides sufficient methionine to support normal protein synthesis
(probably on the order of 10 to 20 micromolar). Where we have needed to
optimize incorporation, we have used serum dialyzed extensively against
0.15 M sodium chloride to remove the endogenous methionine and
reconstituted labeling medium with 2.5 to 5 micromolar methionine. It is
then important to do pilot experiments to determine how long linear
incorporation can be sustained at the cell densities you plan to use
(for very short labeling times incorporation can be increased by using
high concentrations of suspension cells or very small volumes of medium
for attached cells, but you have to check that you are not pushing this
to the extent that you inhibit cell function-- we generally use
linearity and extent of methionine incorporation as a measure of
function-- a simple way to monitor this is to take samples into 0.1 ml 1
N sodium hydroxide, add about 600 micrograms of BSA as carrier, and then
precipitate by adding 4 ml 5% TCA [in a disposable 12 x 75 mm tube]--
filter onto GF/C filter disks, wash tubes and filters with TCA, and
count using a scintillation cocktail that can tolerate water).



More information about the Methods mailing list