Protocol

Reduction and S-Carboxymethylation of Proteins: Microscale Method

This protocol was adapted from "Peptide Mapping and Sequence Analysis of Gel-Resolved Protein," Chapter 7, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Reduction and alkylation of microscale amounts of protein (10-50 µg) require special attention. Classical methods for desalting and dialysis should be avoided due to large sample losses. Adding a small quantity of detergent to the protein solution during all manipulations can also reduce protein loss. This protocol describes a procedure for reduction and S-carboxymethylation of low-microgram amounts of protein. Samples are reduced and alkylated in small volumes (<200 µl) in the presence of detergent and desalted (and concentrated) by RP-HPLC in yields of >90%.