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].
1
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.
2
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
3
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.
4
Section 5. Instructions for Use
General Guidelines . The goal of the first step is to lyse the
cells of interest directly in Reagent 1 using a physical lysis
procedure. The method of lysis will vary greatly depending on
cell type. Follow standard procedures for cell growth and tissue harvesting and standard methods for physical cell lysis.
For example, bacterial cultures can be harvested by centrifu gation then washed in Reagent 1 to remove excess medium.
The cells can then be suspended in Reagent 1 and lysed by
sonication or by liquid shearing, as in a French press. Animal
tissues can be harvested and then homogenized or sonicated
directly in Reagent 1. Plant tissues can be ground in liquid
nitrogen and the powder suspended in Reagent 1 followed by
homogenization if necessary. Tissue culture cells can be
harvested by centrifugation, washed in a medium-free, isotonic buffer and repelleted. It is important to remove all of the
wash buffer from the pellet prior to the addition of Reagent 1.
Sonication or homogenization can be used to achieve
complete lysis. For best 2-D PAGE results, it is generally
worthwhile to treat the homogenates with nucleases. For this,
add a mixture of DNase I and RNase A to final concentrations
of 20–100 µg/ml and 5–25 µg/ml, respectively. Alternatively,
nonspecific endonuclease at 150 units/ml can be used to
degrade nucleic acids present in the mixture.
Use relative amounts of sample cells or tissue and lysis buffer
estimated to yield about 50 mg/ml of protein solution. The
amount of protein in a cell sample can be reasonably
estimated as its dried weight. For example, sonication of a
5
suspension of 0.5 g (wet weight — equivalent to about 100
mg dry weight) of Escherichia coliin 2 ml of Reagent 1results
in effective lysis and fractionation.
Lysis conditions are very important to the success of a
sequential extraction. Proper conditions for thorough lysis
should be determined empirically. For example, overly
aggressive sonication can denature some proteins, while
insufficient sonication can leave some of the cells intact.
Several texts and Internet sites can be consulted for details of
cell culturing and growth and for methods to lyse a wide
variety of cell types. For example, the ExPASy
(http://www.expasy.ch) and Biobase (http://biobase.dk/cgibin/celis) web sites with their associated links contain much
information on sample preparation. Several texts, such as
References 6–9, are valuable sources of information.
Sequential Extraction . Refer to the schematic illustration
for a flow chart of the sequential extraction procedure.
Extraction 1
1
Place the desired starting mass of cells or tissue in a suitable
tube and add Reagent 1 in a ratio estimated to yield at least
50 mg/ml of protein upon lysis.
2
Lyse the cells by sonication or any other suitable physical
means.
3
Transfer the suspension to centrifuge tubes and centrifuge
the sample until firm pellets form. For example, centrifuge E.
coli lysates at top speed in a benchtop microcentrifuge at
room temperature for 10 min.
4
Recover the supernatant and determine its protein content
6
with the Bio-Rad protein assay (Bradford), catalog # 500-0001,
or the modified assay shown below.
5
Wash the insoluble pellet from Step 3 twice with the same
volume of Reagent 1 used in Step 1. It is valuable to
determine the protein concentrations in the washes. Discard
the washes.
6
Store the supernatant frozen until it is used in 2-D PAGE.
7
Dilute the supernatant from Step 6 to standard protein loads
for 2-D PAGE (roughly 1 µg/µl) with extraction solution 2
[Reagent 2 containing a 1:100 dilution (2 mM) of reducing
agent TBP].
8
Because of the ionic nature of the extracts, it is beneficial to
begin the isoelectric focusing run at low voltage. For example,
limit the voltage to 250 V for 1 hr then 500 V for 1 hr before
beginning a ramp up to the final focusing voltage. Use paper
wicks under the electrodes to capture impurities in the
samples.
Extraction 2
1
Prepare extraction solution 2 by making a 1:100 dilution of
reducing agent TBP (to 2 mM) into a quantity of Reagent 2.
Mix 10 µl of reducing agent TBP with each 1 ml of Reagent 2
that is used.
2
Use extraction solution 2 to solubilize proteins in the pellet
from Extraction 1 (Extraction 1, Step 5). Use a volume of
extraction solution 2 that is about half the volume of Reagent 1
used in the first extraction. The best volume to use should be
determined empirically.
7
3
Vortex the mixture for 5 min. For some samples, it may be
necessary to also sonicate the suspension or to aspirate it
through a fine-gauge needle to solubilize the protein.
4
Centrifuge the mixture to give a firm pellet and a clear
supernatant.
5
Recover the supernatant and determine its protein
concentration. The modified Bio-Rad protein assay procedure
shown below is recommended for determining protein
concentrations in extraction solution 2. Store the supernatant
frozen until it is used in 2-D PAGE.
6
Wash the pellet from Step 4 twice with extraction solution 2,
determine the protein concentrations in the washes and
discard them.
7
Dilute the supernatant from Step 5 to standard protein loads
for 2-D PAGE (roughly 1 µg/µl) with extraction solution 2.
8
Because of the ionic nature of the extracts, it is beneficial to
begin the isoelectric focusing run at low voltage. For
example, limit the voltage to 250 V for 1 hr then 500 V for 1
hr before beginning a ramp up to the final focusing voltage.
Use paper wicks under the electrodes to capture impurities in
the samples.
9
Discard unused extraction solution 2.
Extraction 3
1
Prepare extraction solution 3 by making a 1:100 dilution of
reducing agent TBP (to 2 mM) into a quantity of Reagent 3.
Mix 10 µl of reducing agent TBP with each 1 ml of Reagent 3
that is used.
8
2
Use extraction solution 3 to solubilize proteins in the pellet
from Extraction 2. Use approximately the same volume of
extraction solution 3 as was used of extraction solution 2.
Empirically determine the best volume for the third extraction.
3
Vortex and centrifuge as described above.
4
Recover the supernatant and determine its protein content.
The modified Bio-Rad protein assay procedure shown below
is recommended for determining the protein content in
extraction solution 3. Store the supernatant frozen until it is
used in 2-D PAGE. It may be informative to wash the pellet
and determine the protein concentration in the wash solution.
5
The pellet can be extracted with SDS to yield highly insoluble
proteins as described in [1].
6
Dilute the supernatant from Step 4 to standard protein loads
for 2-D PAGE (roughly 1 µg/µl) with extraction solution 3.
7
Because of the ionic nature of the extracts, it is beneficial to
begin the isoelectric focusing run at low voltage. For example,
limit the voltage to 250 V for 1 hr then 500 V for 1 hr before
beginning a ramp to the final focusing voltage. Use paper
wicks under the electrodes to capture impurities in the
samples.
8
Discard unused extraction solution 3.
The three extracts can have different conductivity. With some
isoelectric focusing chambers, best reproducibility is achieved
by focusing the three extracts separately.
9
An example of the sequential extraction of an E. coli
preparation is shown in the figure.
Schematic Illustration of the 3-Step Sequential
Extraction Protoco l
Cell Sample
Step 1
Step 2
Step 3
Reagent 1
Solution 2
Solution 3
Supernatant 1
Insoluble Pellet from
Extraction 1
Supernatant 2
Insoluble Pellet from
Extraction 2
Supernatant 3
Insoluble Pellet from
Extraction 3
10
Solution 2
Gel 1
Gel 2
Gel 3
A
B
C
D
Two-dimensional gel analysis of extracts from E. coli.
E. coli W3110 was collected by centrifugation. The cell pellet was
suspended in Reagent 1 and the cells were lysed by sonication. One
portion of the sonicated cell suspension, containing 200 µg of protein,
was diluted in extraction solution 3. The proteins soluble in solution 3
were separated by 2-D PAGE as a “whole cell extract” (A). Another
portion of the sonicated cell suspension was subjected to the
sequential extraction protocol. (B) 2-D PAGE separation of 200 µg of
protein from Extract 1. (C) 2-D PAGE separation of 200 µg of protein
from Extract 2. (D) 2-D PAGE separation of 200 µg of protein from
Extract 3. First-dimension separation was by isoelectric focusing
using ReadyStrip IPG strips pH 4 to pH 7. The second-dimension
separation was by SDS-PAGE in an 8%T–16%T polyacrylamide
gradient gel.
11
Modified Bio-Rad Protein Assay
This modification to the standard Bio-Rad protein assay
procedure (catalog # 500-0001) is recommended to determine the protein content of Extracts 2 and 3 (modified from
[10] and [11]). It is not necessary with Extracts 1, but it can be
used with Extracts 1, the content of the samples, not the procedures. The high concentrations of detergents and
chaotropes in Reagents 2 and 3 interfere with many other
types of assays.
1
Prepare bovine gamma globulin (BgG) standards at about
14 mg/ml.
• Rehydrate protein standard I with 2 ml of deionized water.
Disregard the instructions with the standard that call
for rehydrating this protein with 20 ml of water. The
protein concentration of standard, when rehydrated
with 2 ml of water, will be 10 times the concentration
shown on the bottle.
2
Prepare a 1:4 dilution of dye reagent concentrate.
• Mix 1 part of the dye reagent with 3 parts of water and
filter the diluted dye through Whatman Number 1 filter
paper, or equivalent.
3.5 ml of diluted dye reagent are used in each standard
and assay tube. Prepare excess dye.
3
Prepare 0.12 N HCl (nominal) by diluting concentrated HCl
1:100 with water.
• Add 10 µl of concentrated HCl to 1 ml of water.
• 80 µl are used in each assay tube. Prepare excess.
12
4
Prepare a standard curve covering the range of 2–280 µg.
• Dilute the BgG standard with extraction solution in a two-
fold (or other) dilution series from 14 to 0.1 mg/ml. TBP can
be omitted from the standard-curve dilutions.
The standard curves generated with gamma globulin in the
three extraction solutions are similar. For approximating
concentrations, it is acceptable to use a single standard
curve generated with the protein dissolved in only one of
the extraction solutions (or in water). For more accurate
determinations, prepare the dilution series in the individual
extraction solutions. The high-concentration standard can
be in water.
• Place 80 µl of 0.12 N HCl (nominal) in each assay tube.
• Mix 20 µl of each dilution of BgG standard with the 80 µl
of 0.12 N HCl (nominal) in each separate assay tube. Mix
gently.
• Add 3.5 ml of diluted dye reagent to each tube. Vortex gently.
• Measure the absorbances at 595 nm (A
• Draw a curve of A
the dilutions of BgG.
versus the amount of protein (µg) for
595
) after 5 minutes.
595
The curve is nonlinear.
5 Assay the extracts in a manner analogous to the preparation
of the standard curve.
• Place 80 µl of 0.12 N HCl (nominal) in each assay tube.
• Mix 20 µl of each extract with the 80 µl of 0.12 N HCl
(nominal) in each assay tube.
13
Very high-concentration extracts, with protein levels
above the upper limit of the standard curve, require that
lesser volumes of them be assayed. In such cases,
increase the amount of 0.12 N HCl (nominal) used so that
the total volume is 100 µl.
• Add 3.5 ml of diluted dye reagent to each tube. Vortex gently.
• Measure the absorbances at 595 nm (A
• Use the standard curve generated in Step 4 to estimate the
concentration of the sample proteins from the A
mea surements.
Sequential Extraction Standard Curve
12
) after 5 minutes.
595
595
1
0%
0.6
0.4
0.2
0
Solution 2
Solution 3
Reagent 1
0 50 100 150 200 250 300
Bovine gamma globulin, in micrograms
Representative Standard Curves. Standard curves
were generated as described above with serial dilutions of
bovine gamma globulin in the three extraction solutions
14
Section 6. References
1
Molloy, MP, et al. Extraction of membrane proteins by differential
solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis. 1998 May;19(5):837-44.
2
Herbert, BR, et al. Improved protein solubility in two-dimensional
electrophoresis using tributyl phosphine as reducing agent.
Electrophoresis, 1998 May;19(5):845-51.
3
Rabilloud, T, et al. Improvement of the solubilization of proteins in
two-dimensional electrophoresis with immobilized pH gradients.
Electrophoresis, 1997 Mar-Apr;18(3-4):307-16.
4
Rabilloud, T. Solubilization of proteins for electrophoretic analyses.
Electrophoresis. 1996 May;17(5):813-29.
5
Herbert, B. Advances in protein solubilisation for two-dimensional
electrophoresis. Electrophoresis, 1999 Apr-May;20(4-5):660-3.
6
Deutscher, M.P., Ed., Guide to Protein Purification. Methods in
Enzymology,182. Academic Press, San Diego (1990).
7
Bollag, DM, Rozycki, MD, and Edelstein, SJ, Protein Methods.
Second Edition. Wiley-Liss, New York (1996).
8
Celis, JE, Ed., Cell Biology. A Laboratory Handbook, Second
Edition. Volumes 1–4. Academic Press, San Diego (1998).
9
Link, AJ, Ed., 2-D Proteome Analysis Protocols. Methods, Mol
Biol. 112. (1999).
10
Ramagli, L. S., and Rodriguez, L. V., Electrophoresis, 6,559–563
(1985).
11
Ramagli, L. S., In 2-D Proteome Analysis Protocols, Methods, Mol
Biol, (A. J. Link, Ed.), 99–103. Humana Press, Totowa, NJ (1999).
15
Section 7. Product Information
Catalog # Product Description
163-2100 ReadyPrep Sequential Extraction Kit
163-2101 Reducing Agent TBP, 200 mM, 0.6 ml
163-2102 ReadyPrep Reagent 1, 1 vial
163-2103 ReadyPrep Reagent 2, 1 vial
163-2104 ReadyPrep Reagent 3, 1 vial
500-0001 Bio-Rad Protein Assay Kit I with Bovine Gamma
500-0005 Protein Standard I, bovine gamma globulin
500-0006 Bio-Rad Protein Assay Dye Reagent Concentrate,
Globulin Standard
450 ml
16
Bio-Rad Laboratories
2000 Alfred Nobel Dr., Hercules, CA 94547
510-741-1000 4006167 Rev B
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