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%.
