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5x met/cys media

5x met/cys media - Protocols and Methods Forum

5x met/cys media - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-10-2007, 08:18 PM
Lisa Imboden
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Default 5x met/cys media



I'm trying to do a pulse-chase experiment to determine the half-life of
my protein. The protocol calls for incubating with met/cys free DMEM,
followed by incubations with [35S]-labeled met/cys, followed by washing
with regular media and chasing with media containing 5X met/cys. I
can't figure out the best way to make media containinng 5X met/cys.
I've read that met and cys are both susceptible to oxidation and
therefore need to be made in degassed water. I can't find any
information about how other people have made their 5X met/cys media.
Any suggestions?

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  #2  
Old 05-10-2007, 09:19 PM
WS
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Default 5x met/cys media

Is said to be available from Gibco/LifeTech and others. Some people
just use normal medium for the chase. Maybe supplementing with Amino
Acid Mix is helpful.

Wo

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  #3  
Old 05-16-2007, 12:54 PM
Dr Engelbert Buxbaum
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Default 5x met/cys media

Am 10.05.2007, 16:18 Uhr, schrieb Lisa Imboden <[Only registered users see links. ]>:


Dissolve the amino acids at, say, 100 x the required concentration in
water and sterile-filter. Note that you may need a counter ion (acid or
base) to get them into solution. The sterile stock solution can then be
added to the medium, like you do with other additives. Degassing the water
is easy. Distilled water is, of course, ipse facto also degassed. Water
purified by ion exchange or reverse osmosis is sonicated a couple of
minutes or stirred while conected to a vacuum pump.
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