Protein Protocols Protein Protocols

SDecS-PAGE of Nucleic Acid Binding Proteins 81
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Identification of Nucleic Acid Binding Proteins
Using Nondenaturing Sodium Decyl Sulfate
Polyacrylamide Gel Electrophoresis
(SDecS-PAGE)
Robert E. Akins and Rocky S. Tuan
1. Introduction
Methods for the identification and characterization of nucleic acid binding proteins,
such as DNA binding transcription factors, typically involve gel retardation assays
(1,2) or Southwestern analysis (3). Gel retardation assays allow the detection of DNAbinding
factors by assessing the degree to which protein binding affects the electrophoretic
mobility of specific DNA sequences. One drawback of gel retardation assays
is that only the presence of the protein is indicated; specific information concerning
protein molecular weight, or other characteristics, is obtained only through additional
methods. Southwestern analysis detects DNA binding proteins through the use of
nucleic acid probes applied to protein blots prepared from sodium dodecyl sulfate
(SDS) gels. The Southwestern technique relies on the limited ability of proteins to
renature after SDS-gel electrophoresis and does not specifically identify binding by
protein complexes, which are dissociated during sample processing.
In this chapter, we describe a novel method for the identification of nucleic acid
binding proteins and protein complexes. The method is based on a nondenaturing gel
electrophoresis system. The system allows the separation of proteins and protein complexes
as a function of log Mr with the maintenance of DNA oligomer binding activity.
The use of the anionic detergent sodium decyl sulfate (SDecS) (see Note 1) in the gel
electrophoresis system allows the identification of specific DNA binding proteins or
protein complexes based on molecular size.
The SDecS-polyacrylamide gel electrophoresis (SDecS-PAGE) technique is
straightforward. In brief, samples solubilized in a SDecS buffer are electrophoresed in
a discontinuous gel (4,5) comprised of acrylamide with SDecS in a Tris-HCl buffer and
using a Tris-glycine running buffer that also contains SDecS. The resulting formulation
is similar to standard sodium dodecyl sulfate (SDS) electrophoresis systems with
SDecS directly substituted for SDS.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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