Hi Jeremiah -- the short answer is Yes You Must! Don't whether you need
that high concentration generally, but if it's not too osmotically
active, the crystals should be fine; test it.
But when mounting SeMet crystals, it's always a good idea to add fresh
DTT or TCEP or whatever, it seems to be rather effective at reversing
oxidation.
(Are you sure it's oxidized? I assume you've checked the crystals by
mass spec?)
phx
Jeremiah Farelli wrote:
Hello all,
I posted here a few months ago. My post dealt with a protein crystal that
was very sensitive to oxidation (with the SeMet derivative even more so).
The protein grows in 24% PEG 1500, no more than 1 mM DTT, 0.05 M Hepes, pH
7.5, 1% glycerol. TCEP will not work, even at very low concentrations.
Does anyone have any experience with soaking the crystals in high
concentrations (10-50 mM?) of DTT or TCEP before freezing? Could this
feasibly be used to "reverse" any protein oxidation right before freezing?
