Biology Reference
In-Depth Information
Chapter 10
Comparative Analyses of Protein Complexes
by Blue Native DIGE
Katrin Peters and Hans-Peter Braun
Abstract
Classically, DIGE is carried out on the basis of two-dimensional (2D) IEF/SDS PAGE. This allows comparative
analyses of large protein sets. However, 2D IEF/SDS PAGE only poorly resolves hydrophobic proteins
and is not compatible with native protein characterizations. Blue native PAGE represents a powerful alter-
native. Combined with CyDye labeling, blue native DIGE offers several useful applications like quantitative
comparison of protein complexes of related protein fractions. Here we present a protocol for fl uorophore
labeling of native protein fractions for separation by blue native PAGE.
Key words: Blue native PAGE, DIGE labeling, Protein complexes, Membrane proteins, Mitochondria
Abbreviations
2D
Two-dimensional
BN PAGE
Blue native polyacrylamide gel electrophoresis
BN/SDS PAGE
Blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis
DIGE Difference gel electrophoresis
DMF Dimethylformamide
IEF/SDS PAGE Isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis
pI
Isoelectric point
Phenylmethylsulfonyl fl uoride
PMSF
1. Introduction
Fluorophore-based difference gel electrophoresis (DIGE) represents
an ideal system for comparative proteomics. Different protein
fractions can be coelectrophoresed on a single gel, thereby elimi-
nating gel-to-gel variations. If evaluated with special software tools,
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