Ian A. York
iayork at panix.com
Thu Mar 13 22:43:49 EST 1997
In article <5g921k$5rm at mserv1.dl.ac.uk>,
L.Kempster <l.kempster at ic.ac.uk> 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.
It depends on your protein and what you're trying to look at. I think one
hour is a good starting point; most proteins will synthesize a detectable
amount in that period, without most of it degrading. If you have a very
short-lived protein, you may need to do shorter labels; if you have a
protein with a long half life, which is synthesized slowly, you may need
to do a multi-hour or overnight label. You need to determine this more or
less empirically, unless you already know something about your protein's
Also, think about the Met/Cys flux. Some cells have large reserves of Met
and Cys, so that you really do need a significant pre-incubation to
deplete that. Others lose it relatively rapidly. Even if you don't
preincubate with Met-free medium, you will still get some incorporation of
the hot stuff; it's just that it must compete for incorporation with the
cold stuff. My own suspicion is that a traditional preincubation with
Met-free medium--which tends to be rather short--doesn't do a lot to
deplete stores in many cell types. I've tried skipping the preincubation
in some cases and often see little if any difference.
In a long (say, >4 or 6 hours) consider supplementing the hot Met with
cold stuff. In a very long label, it's possible for the cells to
incorporate all the hot Met early on (especially if they're metabolically
active and you don't put in a ton of Met). Then you'll get low labelling
at the end of the day, which will *look* like poor incorporation, though
it's the reverse. By supplementing with cold Met--maybe do something like
adding 1/20 volume normal medium--you end up with the cell being able to
synthesize proteins throughout. Even though all of those proteins have
fewer hot Mets compared to cold, at least the protein is actually there
and is detectable.
>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?
FCS does contain some Met and Cys. You can dialyze your FCS, though (and
then filter sterilize it) to provide your cells with growth factors. This
is not particularly essential for most cell types in a short (<30 minutes)
label, but can be helpful for longer labels.
Hope this helps.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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