ReadyPrep™Protein Extraction Kit
(Membrane II)
Instruction Manual
Catalog #163-2084
For technical service, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Section 1 Introduction…………………………………...1–2
Section 2 Kit Specifications…………………………......3–5
Section 3 Storage Conditions………………………..........5
Section 4 Reagent Preparation…………………...........6–7
Section 5 Instructions for Use…………………...........8–13
Section 6 Appendix……………..…………….................14
Section 7 References……………..……………...............15
Section 8 Product Information………………............16–17
Section 1
Introduction
The ReadyPrep protein extraction kit (membrane II) is
designed as a simple, rapid and reproducible method to
isolate cellular membranes and then to extract the
associated integral membrane proteins into a solution
compatible with two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE). The ReadyPrep protein extraction kit (membrane II) joins two existing ReadyPrep kits for
isolation of membrane proteins; it is optimized to effectively
isolate more complex proteins that contain more than two
transmembrane domains, or are resistant to extraction by
the ReadyPrep membrane I kit. The signal kit is specific for
isolation of proteins involved in intracellular membrane
trafficking and signaling pathways. All three of these membrane kits reduce sample complexity using cellular location
as a way to fractionate. The procedure for the ReadyPrep
protein extraction kit (membrane II) is based on the original
work of Fujiki et al.
fractions from rat liver after carbonate extraction and the
later adaption of the technique by Molloy et al.
Proteomic analysis of E. coli outer membrane proteins.
The kit contains all the reagents necessary for membrane
isolation and membrane-protein extraction and the protocol
1
for the isolation of cellular membrane
2
for
1
can be applied to a wide variety of biological samples, from
animal cells and tissues to yeast, bacteria and plant
tissues. After the cells or tissue are disrupted using an
ultrasonic probe, the extract is incubated in a Membrane
Protein Concentrating Reagent. This high pH buffer strips
away proteins weakly-associated with membranes while
leaving membrane fragments with their associated integral
proteins intact. Ultracentrifugation is used to concentrate
the membranes into a pellet, and the proteins are
solubilized from the washed pellet using an isoelectric
focusing-compatible 2-D rehydration/sample buffer. The
2-D Rehydration/Sample Buffer 1 utilized in the kit is a
strongly chaotropic buffer containing the zwitterionic
detergent ASB-14, making this solution one of the most
powerful solubilizing reagents available for 2-D gel
electrophoresis. The kit also contains the reducing agent
tributylphosphine (TBP), which has been shown to be more
efficient than dithiothreitol in keeping proteins reduced in
2-D PAGE applications.
3
2
Section 2
Kit Specifications
Each ReadyPrep protein extraction kit (membrane II)
provides sufficient reagents to perform 10 extractions each
starting with 100–200 mg of cells or tissue. The procedure
can easily be scaled up to accommodate larger amounts of
cells or tissue. The entire procedure can be completed in
less than 3 hr.
Items Supplied With Kit
• 2-D Rehydration/Sample Buffer 1. 2 vials. Lyophilized.
After reconstituting, each vial contains 10 ml of 7 M urea,
2 M thiourea, 1 % (w/v) ASB-14 detergent, 40 mM Tris
base, and 0.001% Bromophenol Blue.
• Lysis Buffer. 5 pouches. Each pouch reconstitutes to
make 50 ml of Lysis Buffer.
• Membrane Protein Concentrating Reagent. 5 pouch-
es. Each pouch reconstitutes to make 125 ml of membrane protein concentrating reagent.
• TBP Reducing Agent. 1 ampoule containing 0.6 ml of
200 mM tributylphosphine (TBP) in 1-methyl2-pyrrolidinone sealed under nitrogen gas.
• Empty Vial. 1 storage vial for TBP reducing agent.
• Instruction Manual, 1.
3
Items Required But Not Provided
• 2.0 ml microcentrifuge tubes (for example, VWR catalog
#20170-237).
• Disposable round-bottom tubes for plant sample
extraction (for example, VWR catalog # 60818-725).
• ReadyPrep 2-D cleanup kit (Bio-Rad catalog #163-2130)
for plant sample extraction. When working with plant leaf
tissue, removal of interfering compounds, such as
phenolics, is essential for high quality 2-D gels with
minimal horizontal streaking.
• 50 ml conical centrifuge tube(s), disposable, (for
example, VWR catalog #20171-030).
• 2 clean 125–150 ml beakers or bottles plus stirring bars.
• Ultracentrifuge, rotor and tubes capable of spinning at
100,000 x g. Typically, tubes should have ~30 ml
capacity.
• Microcentrifuge capable of spinning at 12–16,000 x g
at 4°C.
• Sonicator
• Benzonase (endonuclease) (for example, Sigma
catalog #E1014 or #E8263)
• 1 M MgCl
2
solution
• Protease inhibitor(s) (optional)
• Carrier ampholytes (for example, Bio-Lyte
3/10
ampholyte, Bio-Rad catalog #163-2094)
4
• ReadyPrep proteomic grade water (Bio-Rad catalog
#163-2091)
• RC DC
™
Protein Assay (Bio-Rad catalog #500-0121 or
#500-0122)
Section 3
Storage Conditions
Store the unopened kit at room temperature. After opening,
unused reconstituted ReadyPrep 2-D Rehydration/Sample
Buffer 1 should be aliquoted in 1 to 2 ml volumes and
stored frozen at –80°C. After opening, transfer the
ReadyPrep TBP reducing agent to the empty glass vial
provided and store the vial at –20°C to prevent evaporation
of the TBP. Aliquot and store unused Lysis Buffer and
Membrane Protein Concentrating Reagent at –20°C.
5
Section 4
Reagent Preparation
ReadyPrep 2-D Rehydration/Sample Buffer 1::Add
5.6 ml of ReadyPrep proteomic grade water or similar
quality water to one bottle. Swirl the vial gently until the
contents are completely dissolved. The solution can be
warmed slightly in the palm of the hand or in a water bath
to speed the dissolution process. DO NOT heat the
solution above 25 to 30°C to avoid the formation of
cyanates. Cyanates can react with and modify the proteins
in the sample.
ReadyPrep TBP Reducing Agent. Tributylphosphine
(TBP) has an unpleasant odor and is very volatile. Work
with TBP in a fume hood. Wear a laboratory coat and
gloves when handling the ampoule of TBP reducing agent.
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 empty screw-cap storage
vial provided. Screw the cap of the vial down tightly and
store the vial at –20°C to prevent evaporation of the TBP.
While using, keep the vial of TBP reducing agent on ice.
6
Lysis Buffer. Empty the entire contents of one pouch of
Lysis Buffer into a 50 ml disposable tube. Add 50 ml of
ReadyPrep proteomic grade water or similar quality water
to the tube and mix until the solids are completely
dissolved. Rinse the pouch with a small portion of this
buffer, and return the solution to the 50 ml tube. If desired,
protease inhibitors can be added to a portion of this buffer
just before use.
Membrane Protein Concentrating Reagent. Empty
the entire contents of one pouch of Membrane Protein
Concentrating Reagent into a beaker or bottle containing
125 ml of ReadyPrep proteomic grade water or similar
quality water. To speed up dissolving the solid, add the
contents of the pouch while the water is stirring. Mix until
all the solids are completely dissolved. Rinse the pouch
with a small amount of the solution (10–15 ml) and then
return it to the container. Protease inhibitors can be added
to a portion of this buffer just before use if desired.
7
Section 5
Instructions for Use
Extraction of 100–200 mg of sample.
1. Immediately before performing an extraction, prepare
Lysis Buffer, Membrane Protein Concentrating Reagent
and ReadyPrep 2-D rehydration/sample buffer 1 per
the instructions in Section 4. For a standard extraction
you will need approximately 15 ml of Lysis Buffer,
60 ml of Membrane Protein Concentrating Reagent
and 1 ml of 2-D Rehydration/Sample Buffer 1. Chill
both the Lysis Buffer and the Membrane Protein
Concentrating Reagent on ice for 10–15 min before
proceeding.
2. In a 2.0 ml microcentrifuge tube (on ice), add 1 ml of
Lysis Buffer per 100–200 mg of animal tissue or wet
cell pellet from sources such as cell culture, yeast, or
bacteria. For plant tissue add 2–3 ml of Lysis Buffer
per each gram of tissue in, for example, a disposable
14 ml round-bottom tube. The sample-to-buffer
volume ratio indicated above is only a guide and may
be adjusted depending upon the desired scale of the
preparation and type of sample used.
Insufficient volume of Lysis Buffer may result in poor cell
lysis and incomplete solubilization of proteins.
8
Plant tissue should be ground to a fine powder using a
mortar and pestle in liquid nitrogen before addition of Lysis
Buffer.
3. With the sample on ice, sonicate the suspension with
an ultrasonic probe to disrupt the cells and fragment
the genomic DNA. Sonicate the sample using 30 sec
bursts, typically 3–4 times, or until lysis is complete.
Chill the suspension on ice briefly between each
ultrasonic treatment. If necessary, transfer the extract
to a microcentrifuge tube when this step is complete.
Note: Disruption of cells by sonication is dependent on the
cell type. For example, E. coli requires longer sonication
times than animal cells and tissues. Yeast cell disruption
requires even more vigorous sonication The addition of
glass beads or use of a Bead Beater (BioSpec Products) can
greatly improve cell lysis of these sample types.
3a. Optional. Nucleic acid can be degraded by adding
~150 U of Benzonase and 2 µl/ml of 1 M MgCl
2
to the
extract and incubating the sample at 4–8°C for 20 min
before proceeding to step 4.
4. Centrifuge the tube for 10 min at ~3,000 x g in a
microcentrifuge at 4°C to pellet insoluble material and
unbroken cells.
9
5. Remove and dilute the supernatant directly into a
beaker or bottle containing 60 ml of the ice-cold
Membrane Protein Concentrating Reagent. Stir the
suspension slowly on ice for 60 min.
6. Transfer the sample to ultracentrifuge tubes and
centrifuge at 100,000 x g for 60 min at 4°C to
pellet the membranes and membrane proteins.
7. Remove the tubes from the rotor and mark the
position of the pellet. Carefully decant and discard the
supernatant.
8. Wash each pellet briefly with 3 ml of cold Lysis Buffer.
Do not disturb the pellet when adding the buffer. Allow
the buffer to sit on ice, covering the pellet, for 1–2 min
before decanting (longer incubation times may be
required for unusually large pellets). Typically, the wash
will easily decant without dislodging the pellet.
However, if any of the pellet dislodges or is seen floating, then the sample should be re-centrifuged at
>20,000 x g for 20–30 min at 4°C.
9. Repeat this wash step using 3 ml of Lysis Buffer per
pellet.
10. Let the tube stand on ice for 1 min and remove any
liquid collected at the bottom of the tube by using a
pipet.
10
11. Before performing the extraction of the pellet,
complete the preparation of the 2-D Rehydration/
Sample Buffer 1. Prepare only enough complete
2-D rehydration/sample buffer 1 for the number of
extractions being performed. Each extraction will
require 1.0 ml of the solution. The solution is prepared
by adding 10 µl of ReadyPrep TBP reducing agent and
the appropriate ampholyte to 0.2 % (w/v) to every 1 ml
of reconstituted 2-D Rehydration/Sample Buffer 1. The
ampholyte is chosen to match the pH range of the IPG
strip to be used for 2D analysis, though for most applications, Bio-Rad’s Bio-Lyte 3/10 ampholyte (catalog
#163-2094) can be used. If desired, other additions,
such as protease inhibitors can also be made at this
time.
12. Resuspend the membrane-protein pellet(s) from each
extraction with a total of 1.0–2.0 ml of complete 2-D
rehydration/sample buffer 1. Alternatively, the pellets
can be stored at –80°C for later analysis.
13. Vortex and/or sonicate the sample to completely
solubilize the proteins in the pellet.
Note: If using sonication, care must be exercised to prevent
heating of the sample. The temperature of the sample
should not be allowed to rise above 30
sample becomes too cold, precipitation of the urea and
thiourea can occur. If this happens, gently warm the sample
until the precipitate dissolves before proceeding further.
°C. Similarly, if the
11
14. Centrifuge the tube at maximum speed in a
microcentrifuge (~16,000 x g) for 10–20 min at
18–20°C to pellet any remaining cell debris.
15. Remove and transfer the supernatant (Membrane
Protein fraction) to a clean tube and discard any
residual pellet.
16. Determine the protein concentration of the membrane
protein sample. The Bio-Rad RC DC Protein Assay
(catalog #500-0121 or #500-0122) is recommended
for this measurement. This assay allows for accurate
protein quantitation in the presence of detergents,
reducing agents, and other substances that typically
interfere with other protein assays.
17. An appropriate dilution of the membrane protein
fraction may be necessary before IEF/2-D gel analysis.
Refer to Section 6 for guidelines on selecting the
appropriate protein load and volume of sample needed
for a selected IPG strip. For 2-D separation of the
membrane proteins, it is recommended that 2-D
Rehydration/Sample Buffer 1 containing TBP reducing
agent and ampholytes be used for all dilutions. Using
other rehydration/sample buffers that do not contain
ASB-14 detergent may result in some proteins precipitating out before or during isoelectric focusing.
12
Additional ReadyPrep 2-D rehydration/sample buffer 1
is available and sold separately for this purpose (see
Section 8).
Note: For some applications (for example using basic pH
range IPG strips), the membrane protein sample may
require reduction and alkylation treatment before IEF in
order to achieve optimal 2-D separation. The ReadyPrep
Reduction-Alkylation Kit can be purchased separately for
this purpose (catalog #163-2090).
18. The samples are now ready to be loaded onto IPG
strips. Unused membrane protein extracts should be
stored in aliquots at –80°C.
Note when working with plant leaf samples: for best 2-D
separation results treat the sample using the ReadyPrep
2-D Cleanup Kit (Bio-Rad catalog #163-2130) prior to
performing IEF.
13
Section 6
Appendix
2-D Rehydration/Sample Buffer Volume
Before IEF and 2D gel electrophoresis, the protein sample
may need to be diluted to achieve the desired protein load
for the chosen stain. To best determine the volume of
diluent to use, consider the IPG strip length, the pH
gradient of the IPG strip, and the staining or detection
method. To assist with these calculations, the table that
follows indicates appropriate volumes of 2-D
rehydration/sample buffer needed to rehydrate IPG strips of
specific lengths and the approximate amounts of protein
required for detection using silver stain or Coomassie Blue
G-250 stain.
IPG strip length 7 cm 11 cm 17 cm 18 cm 24 cm
Rehydration 125 µl 185 µl 300 µl 315 µl 410 µl
volume per strip
Protein load– 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg
Silver stain
Protein load– 50–100 µg 100–200 µg 200–400 µg 200–400 µ g 400–800 µg
Coomassie G-250
14
Section 7
References
1. Fujiki Y et al., Isolation of intracellular membranes by means of sodium
carbonate treatment: application to endoplasmic reticulum. J. Cell
Biology 93, 97–102 (1982).
2. Molloy MP et al., Proteomic analysis of the Eschericia coli outer
membrane. Eur. J. Biochem. 267, 2871–2881 (2000).
3. Herbert BR et al., Improved protein solubility in two-dimensional
electrophoresis using tributyl phosphine as reducing agent.
Electrophoresis. 19(5), 845–851 (1998).
15
Section 8
Product Information
Catalog # Description
Sample Preparation Kits
163-2086 ReadyPrep Protein Extraction Kit
(Total Protein), 20 preps
163-2085 ReadyPrep Protein Extraction Kit
(Soluble/Insoluble), 20 preps
163-2084 ReadyPrep Protein Extraction Kit
(Membrane II), 10 preps
163-2130 ReadyPrep 2-D Cleanup Kit, 50 preps
163-2089 ReadyPrep Protein Extraction Kit
(Cytoplasmic/Nuclear), 50 preps
163-2088 ReadyPrep Protein Extraction Kit
(Membrane I), 50 preps
163-2087 ReadyPrep Protein Extraction Kit
(Signal), 50 preps
163-2090 ReadyPrep Reduction-Alkylation Kit,
50 preps
163-2100 ReadyPrep Sequential Extraction Kit,
5–15 preps
16
Rehydration/Sample Buffers
163-2083 ReadyPrep 2-D Rehydration/Sample
Buffer 1, 10 ml, containing 7 M urea, 2M
thiourea, 1% ASB-14, 40 mM Tris base,
and 0.001% Bromophenol Blue
Protein Quantitation Kits (also see bulletin 2610)
500-0121 RC DC Protein Assay Kit I, 500 standard
assays, bovine γ-globulin standard
500-0122 RC DC Protein Assay Kit II, 500 standard
assays, bovine serum albumin standard
Buffer Components
163-2101 Tributylphosphine (TBP), 200 mM, 0.6 ml
163-2094 100X Bio-Lyte 3/10 Ampholyte, 1 ml
163-2091 ReadyPrep Proteomic Grade Water
Coomassie is a trademark of Imperial Chemical Industries, PLC.
Benzonase is a trademark of Merck KGaA.
17
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
(510) 741-1000
1-800-424-6723
4110157 Rev A
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