Cys-Met free media?

Sumio Matzno smatzno at IECR02.IECR.MUKOGAWA-U.AC.JP
Mon Sep 27 05:59:22 EST 1999


Dear all:

We now start to try pulse-chase protein labelling for the evaluation of 
the clearance of intracellular protein in skeletal myoblasts.  Our 
protocol is following:

1. Cells are plated onto 60mm dish.  24hr later medium is changed to DM 
and culture for subsequent 24 hr.

2. Then cells are preincubated with Cys and Met-free medium for 4-6 hrs

3. Replace medium by fresh Cys and Met-free medium with 35S-Cys,Met (0.3 
mCi/mL) and incubate for the time intervals required.

4. Wash cells with PBS, then lyse cells with Tris buffer containing 0.1% 
Triton X-100 and some protein stabilizers.

5. Protein is collected by immunoprecipitation and analyze SDS-PAGE and 
autoradiography.

My problem is how obtaining Cys-Met free medium.  In some catalog 
(Gibco, Flow etc) I can't find Cys-Met free DMEM: only Met-free DMEM is 
found.  Does anyone know where I can purchase Cys-Met free DMEM, or we 
can use only Met-free DMEM for this experiment?

Any help wold be gratefully appreciated.
Regards, 
==============================================================
Sumio Matzno, Ph.D.: First Department of Biochemistry
School of Pharmaceutical Sciences, Mukogawa Women's University
Phone:+81-798-45-9948, Facsimile:+81-798-41-2792
smatzno at mwu.mukogawa-u.ac.jp (lab) smatzno at sannet.ne.jp (home)
http://ph.mukogawa-u.ac.jp/~seika1/
==============================================================




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