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.
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.