Protocol

Concentrating Acrylamide Gel Spots

This protocol was adapted from "Amino- and Carboxy-Terminal Sequence Analysis," Chapter 6 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol describes a method for concentrating gel bands or spots excised from 1D or 2D acrylamide gels. Several replicate protein bands (or spots) are often required to provide sufficient material for sequencing. In these situations, it is necessary to excise several stained protein bands or spots from the gels and pre-concentrate them prior to electroblotting for amino-terminal sequencing or internal sequence analysis following in-gel proteolytic (or chemical) fragmentation. In the method described below, concentration is done by re-electrophoresis of the gel pieces in a simple SDS-PAGE gel poured in a single conventional glass Pasteur pipette.