2-mercaptoethanol and DTT

Kresten kresten at my-deja.com
Fri Jun 4 10:54:54 EST 1999



> when using DTT make sure the pH is 8.0 or above. Apparently cleland
> found
> that the efficacy was 10x at pH 8 cf pH7. Please check this before
> using
> as it is done from memory.

One problem when using both DTT and 2ME for reduction is that the
desired reaction will only run at pH values where sidereactions and
other problems also arise.  In -SH chemistry the thiolate is often the
reactive species so often (if you are below pKa) reactions will run
faster at higher pH-values.

Not too long ago I incubated a 1 mM DTT in a 35 degC waterbath in pH 7.0
KPi and pH 8.5 Tris-HCl, since I was going to make protein reductions at
this temperature. I took out samples and reacted with Ellmans reagent
and calculated number of free thiols pr. DTT molecule as a function of
time.

At pH 7.0 I had 1.8 after 66 hours
At pH 8.5 I had 1 after about 4 hours

So your reaction may run higher at higher pH-values but other problems
could arise.

BTW I had purged the buffers with argon and been rather carefull when
taking out samples.

just a comment
Kresten

--
The address kresten at my-dejanews.com is for spambots only. Please mail me
at LysLeuLeu at crc.dk , transforming the pre at -part into my initials.
Kresten Lindorff Larsen, Dept. Yeast Genetics
Carlsberg Laboratory, Denmark


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