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Western Blotting of Basic Proteins 337
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Protein Blotting of Basic Proteins
Resolved on Acid-Urea-Triton-Polyacrylamide Gels
Geneviève P. Delcuve and James R. Davie
1. Introduction
The electrophoretic resolution of histones on acetic acid-urea-Triton (AUT) polyacrylamide
gels is the method of choice to separate basic proteins, such as histone
variants, modified histone species, and high-mobility group proteins 14 and 17 (1–6
and see Chapters 16 and 17). Basic proteins are resolved in this system on the basis of
their size, charge, and hydrophobicity. In previous studies, we analyzed the abundance
of ubiquitinated histones by resolving the histones on two-dimensional (AUT into
SDS) polyacrylamide gels, followed by their transfer to nitrocellulose membranes, and
immunochemical staining of nitrocellulose membranes with an antiubiquitin antibody
(7–9). However, transfer of the basic proteins directly from the AUT polyacrylamide
gel circumvents the need to run the second-dimension SDS gel and accomplishes the
analysis of several histone samples. We have described a method that efficiently transfers
basic proteins from AUT polyacrylamide gels to nitrocellulose membranes (10).
This method has been used in the immunochemical detection of modified histone,
isoforms, and histone H1 subtypes (6,11–13).
To achieve satisfactory transfer of basic proteins from AUT gels to nitrocellulose,
polyacrylamide gels are submerged in 50 mM acetic acid, and 0.5% SDS (equilibration
buffer 1; 2 × 30 min) to displace the Triton X-100, followed by a 30-min incubation in
a Tris-SDS buffer (equilibration buffer 2). The transfer buffer is an alkaline transfer
buffer (25 mM CAPS, pH 10.0, 20% [v/v] methanol). Szewczyk and Kozloff (14) reported
that alkaline transfer buffers increase the efficiency of transfer of strongly basic proteins
from SDS gels to nitrocellulose membranes. We reasoned that this transfer buffer
would improve the transfer of histones from AUT gels that had been treated with SDS.
2. Materials
Buffers are made from analytical-grade reagents dissolved in double-distilled water.
1. Equilibration buffer 1: 0.575 mL of glacial acetic acid (50 mM), 10 mL of 10% (10 g in
100 mL of water) SDS (0.5%), and water to 200 mL.
2. Equilibration buffer 2: 6.25 mL of 1 M Tris-HCl (62.5 mM), pH 6.8, 23 mL of 10% (w/v)
SDS (2.3%), 5 mL of β-mercaptoethanol (5%), and water to 100 mL. 1 M Tris-HCl: 121 g
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
337