Sandy,
Two more points about acrylamide alkylation of Cys:
7. It would be unfortunate if members of this discussion group were to avoid
trying acrylamide as an alkylating agent because of misconceptions about the
reaction conditions. Historical references about acrylamide alkylation of
Cys typically use unnecessarily high concentrations of acrylamide, high
temperature, long reaction times or some combination of these three factors.
As we were all taught in freshman chemistry, reagent concentration, reaction
time and reaction temperature are critical with respect to driving reactions
to completion and you just need to try a few conditions to find what works
for you.
Locally, acrylamide is the Cys alkylating agent of choice for PVDF-bound
samples. The reaction is carried out to completion using 2M acrylamide in 6M
GuHCl at 50 degrees for 5 minutes. Since this is solid phase chemistry,
cleanup is a matter of briefly washing the strip. Ken Mitchelhill's
pre-electrophoresis Cys alkylation protocol (see electronic archives 5 May,
1999 "Re: hemoglobin denaturation for sds-page") uses 1% acrylamide (about
140mM) at room temperature for 30min to overcome 10mM DTT. No sample cleanup
is needed before loading the gel. And you've got to figure that the
concentration of monomeric acrylamide in an SDS gel is extremely low, but
it's still effective in alkylating Cys.
A good place to start when working out acrylamide alkylation conditions
is to substitute a 10-fold molar excess of acrylamide over the reducing agent
thiol concentration (i.e., 200mM acrylamide for 10mM DTT) into your
alkylation protocol in place of your current alkylating agent. Again,
whatever works for you are the right conditions.
8. Off-the-shelf acrylamide, like a lot of organic reagents, gives annoying
UV artifact peaks when injected onto a C18 column. Locally, injecting a 50ul
sample containing 1M acrylamide onto an 800um ID C18 column gives about 10-20
artifact peaks at 206nm which are comparable in height to about 10ng of
peptide. As a reference, injecting 50ul of 20mM off-the-shelf iodoacetic acid
gives about 5 significant artifact peaks, one of which is a very tall iodine
peak (EEEEEEEEEEE!). It would be really great if someone sold a HPLC-grade
version of acrylamide (nudge, nudge, wink, wink, know what I mean, know what
I mean?).
Overall, acrylamide, like the other Cys alkylating agents, has its own
set of conveniences and its own set of problems. And acrylamide is very
convenient to use. Whether it makes sense for you to use acrylamide will be
determined by the convenience/problem tradeoff for your application relative
to your current alkylating agent's convenience/problem ratio.
Hope that helps,
Steve Tindall
Argo BioAnalytica Inc.
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