Protein Protocols Protein Protocols

Electroelution of Proteins 299
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Electroelution of Proteins from Polyacrylamide Gels
Paul Jenö and Martin Horst
1. Introduction
Understanding the function of proteins requires determination of their structures.
Advances in chemical technology make it possible to obtain a picture of global protein
expression in model organisms such as the yeast Saccharomyces cerevisiae. Instrumental
to these breakthroughs was the development of high-resolution two-dimensional
gel electrophoresis and mass spectrometric means for rapid detection and analysis of
peptides and proteins. While sequence information on single spots separated by
two-dimensional electrophoresis can be quickly obtained by today’s technology, structural
characterization of proteins expressed in low abundance is still a difficult task.
For example, in our attempts to isolate components of the import machinery of yeast
mitochondria, some proteins turned out to be expressed at extremely low levels, and it
soon became evident that conventional purification techniques were impractical to
obtain these proteins in amounts sufficient for structural analysis. Furthermore, some
proteins of the mitochondrial import machinery were rapidly degraded by proteases,
and they could be obtained intact only by first denaturing mitochondria with trichloroacetic
acid and then solubilizing them in boiling sample buffer for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1). This in turn made the
separation of several milligrams of denatured proteins necessary, which was best
achieved by preparative SDS-PAGE followed by electroelution of individual proteins.
Proteins isolated by electroelution usually contain large amounts of salts and SDS,
which interfere with enzymatic digestion or Amino-D (N)-terminal sequencing. A number
of methods are available to remove SDS from proteins, including precipitation of
the detergent by organic solvents (2,3), and solvent extraction with (4) or without
ion-pairing reagents (5). However, they all suffer from the disadvantage that once the
detergent is removed, many proteins become virtually insoluble in buffers lacking SDS.
Alternatively, chromatographic methods can be used to remove SDS from proteins.
Simpson et al. (6) described desalting of electroeluates based on the finding that certain
reverse-phase matrices retain proteins at high organic modifier concentrations, whereas
small molecular weight compounds are not. This allows bulk separation of salts, SDS,
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
299