An IonSource Tutorial
Monograph 0004 
Method for the S-Carboxymethylation of Cysteine
.
.
.
..

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)

  1. Reconstitute 1 mg of protein in 1 mL of 100 mM Tris pH 8.5

  2. Add 10 L 1M dithiothreitol and let the reduction proceed for 4hrs ambient or for a shorter time at 37C, (60 min.).  The reduction time will need to be determined experimentally.  These times and concentrations will generally work.

  3. If the sample was heated for the reduction, let it cool to ambient.

  4. Add 25 L of 1M iodoacetic acid,  mix, and let stand for 30 min ambient, in the dark.

  5. Add 20 L 1M dithiothreitol to quench the iodoacetic acid.

  6. Purify the RCMed protein from this matrix using dialysis, spin columns, or a fast reverse phase separation.

  7. The protein is ready for enzymatic digest. 

 


Dithiothreitol


Iodoacetic Acid

 


Cysteine


S-Carboxymethylated 
Cysteine

.

  TOC       Introduction        Reaction       Method       Method Notes            Why Reduce?      RCM Compound Data Base       Order Reagents         References     

 

-
-

home | disclaimer
Copyright 2000-2016  IonSource, LLC  All rights reserved. 
Last updated:  Tuesday, January 19, 2016 02:49:30 PM