Metabolic labelling

John Ladasky ladasky at leland.Stanford.EDU
Thu Mar 13 22:31:16 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.

	I don't know whether it depends on the particular proteins/cells you
are labeling.  I am studying class I MHC, and I label PBMC or cell lines for
four hours.  Once I decided to try overnight labeling (15 hours), and I actu-
ally got nothing!  Not increased labeling, nor increased background, but no-
thing.

>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?

	The primary item supplied by fetal calf serum that is missing from
nutrient medium is growth factors.  You don't need to worry about whether
your cells can *divide* during the labelling step, just that they stay 
alive.  Unless the particular cells you are labelling undergo apoptosis
immediately (within the labelling time) upon withdrawal of serum (which
would be very unusual), you should be fine without FCS.

>Thanks in advance,
>
>Lee Kempster
>Imperial College


-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green



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