Protein Protocols Protein Protocols

SDS-PAGE Mapping 553
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Peptide Mapping by Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis
Ralph C. Judd
1. Introduction
The comparison of the primary structure of proteins is an important facet in the
characterization of families of proteins from the same organism, similar proteins from
different organisms, and cloned gene products. There are many methods available to
establish the sequence similarities of proteins. A relatively uncomplicated approach is
to compare the peptide fragments of proteins generated by enzymatic or chemical cleavage,
i.e., peptide mapping. The similarity or dissimilarity of the resultant peptides reflects
the similarity or dissimilarity of the parent proteins.
One reliable method of peptide mapping is to separate peptide fragments by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Comparison of the
separation patterns reveals the structural relationship of the proteins. Moderate separation
of peptides can be accomplished using this procedure. The technique, first described
by Cleveland et al. (1), is simple, inexpensive, requires no special equipment, and can
be combined with Western blotting to locate epitopes (i.e., epitope mapping [1–3]) or
blotting to nylon membranes for microsequencing (4). Microgram amounts of peptide
fragments can be visualized by in-gel staining, making this system fairly sensitive.
Sensitivity can be greatly enhanced by radiolabeling (see Chapter 78).
2. Materials
2.1. SDS-PAGE
1. SDS-PAGE gel apparatus and power pack (e.g., EC 500, EC Apparatus, Inc., St. Petersburg,
FL, or equivalent).
2. SDS-PAGE Solubilization buffer: 2 mL 10% SDS (w/v) in H2O, 1.0 mL glycerol,
0.625 mL 1 M Tris-HCl, pH 6.8, 6 mL H2O, bromophenol blue to color.
3. Enzyme buffer for Cleveland et al. (1) “in-gel” digestion: 1% SDS, 1 mM EDTA, 1%
glycerol, 0.1 M Tris-HCl, pH 6.8.
4. All buffers and acrylamide solutions necessary for running SDS-PAGE (see Chapter 11).
5. Ethanol: 1 M Tris-HCl, pH 6.8 (50:50; [v/v]).
6. Fixer/destainer: 7% acetic acid, 25% isopropanol in H2O (v/v/v).
7. Coomassie brilliant blue: 1% in fixer/destainer (w/v).
8. Mol-wt markers, e.g., low-mol-wt kit (Bio-Rad, Hercules, CA, or equivalent) or peptide
mol-wt markers (Pharmacia, Piscataway, NJ, or equivalent).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
553