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Standard reduction and S-carboxymethyation
reaction used for proteins and peptides.
Reagents:
1M Dithiothreitol
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 Procedure:
RCM 1 milligram of protein (conc. 1mg/mL)
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Reconstitute 1 mg of protein in 1 mL
of 100 mM Tris pH 8.5
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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
work.
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If the sample was heated for the
reduction, let it cool to ambient.
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Add 25 µL of 1M iodoacetic acid, mix, and
let stand for 30 min ambient, in the dark.
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Add 20 µL 1M dithiothreitol to
quench the iodoacetic acid.
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Purify the RCMed protein from this
matrix using dialysis, spin columns, or a fast reverse phase
separation.
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The protein is ready for enzymatic
digest.
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Dithiothreitol
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