TCEP is often sold as hydrochloride salt and therefore reacts highly
acidic. We usually titrate the concentrated solution *before* we add
it to our proteins. This can be done within small volumes by dropwise
addition of a saturated TRIS solution, check pH by pipetting a drop on
a pH test strip.
Clemens
Zitat von "John A. Newitt" <[EMAIL PROTECTED]>:
At 5:04 PM -0600 11/25/08, Jacob Keller wrote:
Sorry for this very prosaic question:
does anybody have a reference describing the mechanisms of reducing
agents (TCEP, DTT, BME, etc.), and in particular the effects of the
reactions on pH? I think I can draw a convincing reaction mechanism
for myself, but I am not sure theoretically why (or even whether)
the pH drops in the presence of reducing agents (although I am
pretty sure empirically that it does.) I have looked around a bit,
but perhaps have not hit on the right google search terms...
This doesn't directly address the question I think you are asking,
but one obvious way that adding reducing agents could affect the pH
is if they contain significant amounts of strong acids. One vendor
of TCEP that we used in the past presumably had so much residual HCl
in the solid reagent that even when TCEP was added at 1 mM it caused
a significant decrease in pH of a buffered solution. It taught us to
check the pH after adding reducing agents and also to find another
vendor.
- John
