N-ethylmaleimide handling - (Jun/26/2006 )
Hi everybody. Does anybody here use N-ethylmaleimide (for stabilization of SUMO conjugates)? How do you normally use it? Ethanol stock, fresh powder directly into batch of lysis buffer,...? Which concentration (I´ve seen 1mM to 20mM for very similar approaches!!)? Data sheet from Sigma recomends dilution to 50mg/ml (aprox. 400mM) in ethanol for storage, but that means 400mM, just 20x stock in ethanol for some protocols. Is that correct?
Thanks in advance!
Miguelon
How does it exactly stabilize SUMO-conjugates? Does it work also for Nedd8-conjugates?
It´s supposed to inhibit, as part of its broad enzime inhibitor role, specific sterases that very efficiently would cleave SUMO from the target protein in native conditions.
I don´t know whether it works for "Nedd"ylation studies as well, but that inhibitor effect of NEM is supposed to be broad. Is Nedd8 conjugation as labile as SUMOylation?
Thanks, I think it should interfere also with the de-Neddylation of proteins.
hello,
NEM acts to stabilize SUMO conjugates by covalently modifying the sulfhydryl group of the catalytic cysteine on SUMO-specific proteases (as well as pretty much any other reduced cysteine it has access to).
as for preparing it, I've always just made a fresh 1M stock (no need to make a bunch) in 95% ethanol for each exctraction. various groups use different concentrations of NEM in their buffers (ranging from 2 to 20mM). i've had good luck with 20mM.
if you're going to perform mass spec analysis on your NEM-treated samples, be sure to let include that in the searches, as some of your Cys residues will be alkylated w/ NEM and others w/ IAA.
NEM acts to stabilize SUMO conjugates by covalently modifying the sulfhydryl group of the catalytic cysteine on SUMO-specific proteases (as well as pretty much any other reduced cysteine it has access to).
as for preparing it, I've always just made a fresh 1M stock (no need to make a bunch) in 95% ethanol for each exctraction. various groups use different concentrations of NEM in their buffers (ranging from 2 to 20mM). i've had good luck with 20mM.
if you're going to perform mass spec analysis on your NEM-treated samples, be sure to let include that in the searches, as some of your Cys residues will be alkylated w/ NEM and others w/ IAA.
Thanks!!