Bio-Rad ReadyPrep Sequential Extraction Kit User Manual

ReadyPrep

™

Sequential Extraction Kit

Instruction Manual

Catalog #
163-2100

For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD

(1-800-424-6723)

Table of Contents

Section 1. Introduction...........................................1

Section 2. Kit Components.....................................2

Section 3. Storage..................................................3

Section 4. Reagent Preparation..............................3

Section 5. Instructions for Use...............................5

Section 6. References..........................................15

Section 7. Product Information ............................16

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 low­abundance 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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Sequential extraction can be carried out in a single
microcentrifuge tube, minimizing protein loss. The kit is
particularly well suited for use with 2-D PAGE systems
employing immobilized pH gradients for the first-dimension
IEF.

Section 2. Kit Components

Reagent 1. One vial. Lyophilized. Each vial of rehydrated
Reagent 1 contains 50 ml of 40 mM Tris base.

Reagent 2. Three vials. Lyophilized. Each vial of rehydrated
Reagent 2 contains 10 ml of 8 M urea, 4% (w/v) CHAPS, 40
mM Tris, and 0.2% (w/v) Bio-Lyte 3/10 ampholyte.

Reagent 3. Two vials. Lyophilized. Each vial of rehydrated
Reagent 3 contains 10 ml of 5 M urea, 2 M thiourea, 2% (w/v)
CHAPS, 2% (w/v) SB 3-10, 40 mM Tris, and 0.2% (w/v) Bio­Lyte 3/10 ampholyte.

Reducing Agent TBP . One ampoule containing 0.6 ml of
200 mM tributyl phosphine (TBP) in 1-methyl-2-pyrrolidinone
(NMP) sealed under nitrogen gas.

Empty Vial. One storage vial for reducing agent TBP.
Instruction Manual.

Note: CHAPS is 3-[(3-Cholamidopropyl)dimethylammonio]-1-

propanesulfonate, a zwitterionic detergent.
SB 3-10 is N-decyl-N,N-dimethyl-3-ammonio-1-

propanesulfonate, or caprylyl sulfobetaine, a zwitterionic
detergent.

Bio-Lyte 3/10 is a mixture of carrier ampholytes, pH 3–10.

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Section 3. Storage

Store the lyophilized contents of the kit, Reagents 1, 2, and 3,
unopened at room temperature.

Store the sealed ampoule of reducing agent TBP unopened
at room temperature.

Section 4. Reagent Preparation

Reagent 1: Add 50 ml of deionized water to the vial of
Reagent 1. Swirl the vial gently at intervals until the contents
are completely dissolved. Reconstituted Reagent 1 consists
of 50 ml of 40 mM Tris base per vial.

Reagent 2: Add 6 ml of deionized water to each vial of
Reagent 2. Swirl the vials gently at intervals until the contents
are completely dissolved. It will take some time for the
contents of the vial to dissolve. The vial will chill as the urea in
the solids dissolves. Rehydrated Reagent 2 consists of 10 ml
of 8 M urea, 4% (w/v) CHAPS, 40 mM Tris, and 0.2% (w/v)
Bio-Lyte 3/10 per vial.

Reagent 3: Add 6.3 ml of deionized water to each vial of
Reagent 3. Swirl the vials gently at intervals until the contents
are completely dissolved. It will take some time for the
contents of the vials to dissolve. The vial will be chilled as the
urea and thiourea in the solids dissolve. The SB 3-10
detergent will not dissolve completely until the solution has
been warmed slightly. It is often sufficient to roll a vial of
Reagent 3 between the hands for a short period of time to
dissolve the SB 3-10. In some situations, it might be

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necessary to hold the vial in a bath of warm (tap) water for
more efficient warming. Rehydrated Reagent 3 consists of 10
ml of 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v) SB
3-10, 40 mM Tris, and 0.2% (w/v) Bio-Lyte 3/10 per vial.

Storage of rehydrated solution 1, 2, 3. Following
rehydration, aliquot the solutions as convenient and store the
aliquots at -20 °C or lower. A convenient aliquot size is 1 ml,
each placed into a microcentrifuge tube for storage. Thaw the
frozen aliquots prior to use and discard unused portions of
each aliquot. It may be necessary to warm the solution of
Reagent 3 to disperse the SB 3-10 detergent.

Reducing Agent TBP . Tributyl phosphine (TBP) has an
unpleasant odor and is very volatile. Work with it in a fume
hood. Wear a laboratory coat and gloves when handling the
ampoule of tributyl phosphine. Change gloves often. Wipe up
spills with wet towels. Open the ampoule by snapping the top
off at the scored neck. Transfer the entire contents of the
ampoule to the screw-top vial provided. Screw the cap of the
vial down tightly and store the vial at -20 °C or lower to
prevent evaporation of the TBP.

Extraction Solutions. Before carrying out an extraction,
mix 10 µl of TBP solution with every 1 ml of Reagent 2 and
(or) Reagent 3 to be used in the extraction (1:100 dilution).
TBP is not used with Reagent 1. Use only polypropylene or
glass pipettes when distributing the reducing agent TBP
concentrate. Most standard pipette tips, such as those sold
by Bio-Rad, are made of polypropylene. Do not use
polystyrene pipettes, since they will dissolve in the NMP solvent.

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