Standard reduction and S-carboxymethyation
reaction used for proteins and peptides.
1M Iodoacetic acid made up in 1M NaOH
100 mM Tris pH 8.5 or 100 mM ammonium bicarbonate (Note: There is
no need to adjust the pH of ammonium bicarbonate. The Tris base
solution needs to be titrated with concentrated HCL.)
RCM 1 milligram of protein (conc. 1mg/mL)
Reconstitute 1 mg of protein in 1 mL
of 100 mM Tris pH 8.5
Add 10 µL 1M dithiothreitol and let
the reduction proceed for 4hrs ambient or for a shorter time at
37°C, (60 min.). The reduction time will need to be determined
experimentally. These times and concentrations will generally
If the sample was heated for the
reduction, let it cool to ambient.
Add 25 µL of 1M iodoacetic acid, mix, and
let stand for 30 min ambient, in the dark.
Add 20 µL 1M dithiothreitol to
quench the iodoacetic acid.
Purify the RCMed protein from this
matrix using dialysis, spin columns, or a fast reverse phase
The protein is ready for enzymatic