Biology Reference
In-Depth Information
Chapter 26
Application of 2D DIGE in Animal Proteomics
Ingrid Miller
Abstract
Two-dimensional electrophoresis (2 DE) is one of the most important proteomic tools and allows studying
the complexity of proteomes of different origin. This chapter describes a setup for 2D DIGE with minimal
labeling for qualitative and quantitative applications. It relies on homemade gels of medium size and in our
hands has been found useful for a wide variety of separation problems involving complex protein mixtures
of animal or human origin. The basic method is given for serum proteins of different species, but with
minor modifi cations the method may be easily adapted to other sample materials (other body fl uids, cells,
tissues), conditions, or size. Examples are given for simple pattern comparisons (e.g., quality control, fast
comparison of just two samples) as well as for quantitative applications to larger sample sets.
Key words: 2D DIGE, Animal body fl uids, Animal sera, Animal tissues, Homemade IPGs
1. Introduction
Two-dimensional electrophoresis (2 DE) is one of the key methods
to study the proteomes of complex biological fl uids and tissues.
It relies on isoelectric focusing (IEF) under reducing and denatur-
ing conditions in the fi rst dimension and reducing SDS-PAGE in
the second. This combination allows seeing single proteins and/or
protein subunits, separated according to their isoelectric points and
molecular masses and with a very high resolution ( 1 ). Especially in
its early days, 2 DE has suffered from the fact that each sample is
separated on a single gel, and gel-to-gel variation often makes
pattern comparison between gels diffi cult. The new variety of 2D
DIGE helps to minimize this problem and thus allows detecting
more reliably small concentration changes between samples. Protein
detection relies on pre-electrophoretic minimal labeling of the
proteins via (some of their) lysine residues by fl uorescent CyDyes,
Search WWH ::




Custom Search