Help with orthophosphate labeling!
yip at rascal.med.harvard.edu
Fri Aug 22 13:16:25 EST 1997
32P in vivo labelling is very tricky and there are many parameters to
consider. If your protein is abundant, I would choose a shorter
incubation time (say less than 3 hours). If it is rare, I would lower
the specific activity to allow sufficient ATP for cells to survive a
longer incubation time. Pre-incubation to clear the intrinsic pool of
ATP is not necessary since ATP turnover in the cells are very rapid. It
is generally assumed that a high specific activity is required. I used
to use 2 mCi per ml of labelling media. You better hope you get
labelling with that otherwise you gotta repeat the experiment (hence
repeat radioactive exposure).
I've have some limited success with 32P in vivo labelling techniques but
all that I've learnt I got from Current Protocols in Molecular Biology
(the red books). There's a chapter in 32P in vivo labelling explaining
all the parameters to consider and even helpful buffers and reagents.
> I'm trying to label a protein in vivo with orthophosphate. Results are
> not great so far. I've done a 7 hours incubation with 8 mCi (0.8
> mCi/ml), with something like 10 millions NIH 3T3 cells (around 1 mg of
> protein at the end), followed by IP and 5 washes with the extraction
> buffer. Still, I got a lot of background and a very weak band.
> Is it worth trying a longer incubation with 32P, or starting with more
> cells? How can I remove the background?
> Any idea would be very helpful!
> Olivier Coqueret. Ph. D.
> Molecular Oncology Group
> McGill University, Royal Victoria Hospital
> 687 Pine Av. West
> Montreal, Quebec
> Canada H3A 1A1
> Phone: (514) 842 12 31 ex: 5832
> Olivier at lan1.molonc.mcgill.ca
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