Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 3)

Section 1. Introduction
The ReadyPrep sequential extraction kit provides the
reagents necessary to extract proteins of differing solubility
from cell lysates in a form suitable for two-dimensional
polyacrylamide gel electrophoresis (2-D PAGE). It is based on
the work of Molloy et al. [1] and Herbert et al. [2]. Each of the
three solutions in this kit solubilizes a different set of proteins.
Reagent 1 extracts only the most soluble proteins, such as
cytosolic proteins. Reagent 2 is used to extract proteins of
intermediate solubility, while Reagent 3 extracts proteins
otherwise insoluble in Reagents 1 and 2. Some proteins
partition into two or more fractions and others remain
insoluble in the reagents used in this kit.
The extraction solutions can be used independently or
sequentially. Sequential extraction provides a third dimension
of separation based on solubility. It results in manageable 2-D
PAGE protein patterns for simplified protein identification and
gel matching. In addition, the high protein loads obtainable
with the individual fractions enable visualization of low-
abundance proteins.
The three solutions of the ReadyPrep sequential extraction kit
differ in their detergent and chaotrope concentrations [3]. The
insoluble residues from each extraction step are further
extracted with reagents of increasingly stronger solubilizing
power. The reducing agent used in the extractions, tributyl
phosphine (TBP), is more efficient than the commonly used
dithiothreitol, especially in 2-D PAGE applications [4, 5].
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