ReadyPrep
™
Reduction-Alkylation Kit
Instruction Manual
Catalog #163-2090
For technical service, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
(510) 741-1000
1-800-424-6723
4110063 Rev A
Table of Contents
Section 1 Introduction……………………………………...1
Section 2 Kit Specifications…………………………......2–3
Section 3 Storage Conditions………………………..........3
Section 4 Instructions for Use………………….............4–6
Section 5 Appendix……………..……………..............7–11
Section 6 References…………..……………..................12
Section 7 Product Information………………….........13–14
Section 1
Introduction
The ReadyPrep reduction-alkylation kit offers an easy and
effective way of improving spot resolution during 2-D
electrophoresis. This general-purpose kit minimizes streaking and aberrant spot migration by reducing and irreversibly
blocking the formation of inter- and intramolecular disulfide
bonds prior to isoelectric focusing (IEF). Disulfide bond
formation is particularly problematic for basic proteins
because of the increased rate of their formation in an
alkaline environment. Moreover, many reducing agents
become negatively charged during isoelectric focusing and
migrate off the IPG strip, thus allowing disulfide bonds to
re-form (Görg 2000, Hoving 2002). The ReadyPrep
reduction-alkylation kit eliminates the potential for disulfide
bond formation during IEF by first reducing the disulfide
bonds with tributylphosphine (TBP), a powerful nonionic
reducing agent, and then alkylating the sulfhydryl groups
with iodoacetamide (Herbert et al. 2001). This treatment
produces a spot pattern with more spots, fewer streaks,
and greater reproducibility. The entire process can be
completed directly in the rehydration/sample buffer in <2 hr
with very little hands-on time. This kit is compatible with
other ReadyPrep sample preparation kits.
1
Section 2
Kit Specifications
The components provided in the ReadyPrep reductionalkylation kit are listed below. Each kit contains sufficient
reagent to perform a reduction-alkylation reaction on
100 samples of 300 µl each containing up to 10 mg/ml
of protein.
Items Supplied With Kit
3 ampoules TBP (200 mM), 0.6 ml
3 empty glass vials for storage of TBP after opening the
ampoules
5 amber vials iodoacetamide, 56 mg
1 tube alkylation buffer, 2 ml
Caution!
Care should be exercised when handling the components.
TBP should be used only in areas with good ventilation.
Avoid breathing vapor. Avoid contact with skin. In case of
contact with eyes, flush with water and contact a
physician.
MSDS forms are available upon request.
2
Items Required But Not Provided:
• 1.5 ml microcentrifuge tubes
• Microcentrifuge capable of spinning at 12,000–16,000 x g
• Rehydration/sample buffer
Section 3
Storage Conditions
Store the unopened kit at room temperature. Once
opened, follow the storage instructions provided on each
component.
Once an ampoule of TBP has been opened, transfer the
TBP to one of the empty glass vials provided and store
at -20°C.
Iodoacetamide powder should be stored in the dark and is
unstable once in solution. Discard unused iodoacetamide
solution.
3
Section 4
Instructions for Use
Per 500 µl sample volume:
Note: This protocol can be scaled for smaller and larger
protein sample volumes by adjusting the amounts of
alkylation buffer, TBP, and iodoacetamide solution
accordingly.
1. Prepare the iodoacetamide solution immediately before
use by dissolving the contents of one vial in 0.6 ml of
ultrapure water. Bio-Rad ReadyPrep proteomic grade
water (catalog #163-2091) is recommended. If not
prepared previously, open an ampoule of TBP and
transfer the solution into one of the empty glass vials
provided. Keep both solutions on ice.
Note: The iodoacetamide is unstable in solution and
therefore requires preparation immediately before use.
2. Prepare a protein sample in the desired rehydration/
sample buffer (refer to Section 5.1 for recommendations on buffer recipes and volumes). The reductionalkylation reaction can be performed on a
concentrated protein sample (up to 10 mg/ml) and
diluted later with additional rehydration/sample buffer
for loading onto IPG strips.
4
For best results, the protein concentration of the
sample should be determined. We recommend the
™
Bio-Rad RC DC
Protein Assay for protein quatitation
because the assay is suitable for the widest variety of
samples and works in the presence of detergents,
reducing agents, and other agents that interfere with
other protein assays.
Note: The protein sample must be free of reducing agent
(i.e., TBP, DTT, or TCEP) before the reduction-alkylation
reaction is performed. This ensures the necessary molar
excess of iodoacetamide to reducing agent during the
reaction. Additionally, the sample must have a low salt
concentration (<40 mM) after the reduction-alkylation
reaction is performed to maintain low conductivity during
IEF. The reduction-alkylation reaction itself contributes salt
to the sample (30 mM). Therefore, it may be necessary to
remove excess salt prior to IEF. (Alternatively, the
reduction-alkylation reaction can be performed in a higher
protein and salt concentration and subsequently diluted for
IEF.) The ReadyPrep 2-D cleanup kit (catalog #163-2130)
can be used to remove excess reducing agent, salt, and
detergent from the sample, and to concentrate the protein
in the sample, if desired.
3. Adjust the pH of the sample to between 8.0 and 9.0
by adding 15 µl of the alkylation buffer. Mix well by
vortexing.
4. Add 12.5 µl of TBP for a final concentration of 5 mM.
Mix well by vortexing. Incubate at room temperature
for 30 min.
5
5. Add 15 µl of 0.5 M iodoacetamide for a final
concentration of 15 mM. Mix well by vortexing.
Incubate at room temperature for 1 hr.
Note: Longer incubation times may cause spurious
reactions between the iodoacetamide and the protein that
could create artifacts in mass spectrometry analysis.
6. Add an additional 12.5 µl of TBP to quench any
unreacted iodoacetamide. Mix well by vortexing.
Incubate at room temperature for 15 min.
7. Centrifuge at 12,000–16,000 x g for 5 min at room
temperature to pellet any insoluble material.
8. Dilute the protein sample to the appropriate final
volume in rehydration/sample buffer and load onto the
IPG strip. Refer to Section 5.1 to determine the
volumes of rehydration/sample buffer and amounts of
protein needed for different strip lengths and staining
methods. For further improvement of spot resolution,
especially for basic strips (pH 7–10), cup loading is
required. For information on cup loading, see the Cup
Loading Tray instruction manual (4006216) and bulletin
2812.
6
Section 5
Appendix
5.1 2-D Rehydration/Sample Buffer Volume
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. When loading the protein onto the IPG strips
by cup loading, refer to the Cup Loading Tray instruction
manual (4006216) for detailed instructions. When loading
the protein onto the IPG strips by passive/active rehyrdration, refer to the ReadyStrip
(4006166) for detailed instructions.
IPG strip length 7 cm 11 cm 17 cm 18 cm 24 cm
Rehydration 135 µl 200 µl 330 µl 345 µl 450 µl
volume-cup loading
Rehydration 125 µl 185 µl 300 µl 315 µl 410 µl
volume–passive/
active rehyrdration
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 µg 400–800 µg
Coomassie G-250
™
IPG Strip instruction manual
7
5.2 Preparation of 2-D Rehydration/Sample
Buffers
The 2-D rehydration/sample buffer is not a component of
this kit, but is necessary for performing 2-D electrophoresis.
It is not provided in the kit because different protein
samples can require different rehydration/sample buffers.
For convenience, a selection of 2-D rehydration/sample
buffer formulas are provided below.
Do not add reducing agent (i.e. TBP, DTT or TCEP)
to the rehydration/sample buffer before performing the reduction-alkylation reaction. A molar
excess of alkylating agent to reducing agent is
required to ensure efficient alkylation.
Users of the ReadyPrep protein extraction kit (membrane I),
catalog #163-2088, or the ReadyPrep protein extraction kit
(cytoplasmic/nuclear), catalog #163-2089, or the
ReadyPrep protein extraction kit (signal), catalog
#163-2087, should use the protein solubilization buffer
(PSB) and PSB diluent provided with these kits to solubilize
the protein samples. PSB is a proprietary strongly
chaotropic 2-D rehydration/sample buffer (refer to Section
5.2.2.2 for preparation instructions).
8
5.2.1. General-Purpose 2-D Rehydration/Sample
Buffer (no reducing agent)
(8 M urea, 2% CHAPS, 0.2% Bio-Lyte
®
3/10 ampholyte,
0.002% bromophenol blue)
Component Final Concentration Amount to Make 2 ml
Urea (FW 60.06) 8 M 0.96 g
CHAPS 2% (w/v) 0.04 g
100X Bio-Lyte 0.2% (w/v) 20 µl
3/10 ampholyte
Bromophenol Blue 0.002% (w/v) 4 µl of a 1%
Proteomic grade 1.25 ml
water
**
Use an ampholyte buffer that corresponds to the pH range of the IEF
separation to be performed. For example, ReadyStrip
with ReadyStrip micro-range IPG strips and ReadyStrip 7-10 buffer with
ReadyStrip pH 7-10 IPG strips. Bio-Lyte 3/10 ampholyte can be used with
all other ReadyStrip IPG strip pH ranges.
**
(w/v) solution
™
micro-range buffers
5.2.2. Strongly Chaotropic 2-D Rehydration/
Sample Buffers for Highly Hydrophobic Proteins
5.2.2.1. Standard Strongly Chaotropic Buffer
(no reducing agent)
(7 M urea, 2 M thiourea, 4% CHAPS, 0.2% Bio-Lyte 3/10
ampholyte, 0.002% bromophenol blue)
9
Component Final Concentration Amount to Make 2 ml
Urea (FW 60.06) 7 M 0.84 g
Thiourea 2 M 0.304 g
CHAPS* 4% (w/v) 0.08 g
100X Bio-Lyte 0.2% (w/v) 20 µl
3/10 ampholyte
Bromophenol Blue 0.002% (w/v) 4 µl of a 1%
Proteomic grade 1.1 ml
water
*Other neutral or zwitterionic detergents can also be used at concentrations
of 1% to 2% (w/v) to improve solubilization of membrane and hydrophobic
proteins. Examples are n-octyl-β-D-glucopyranoside, SB3-10 (N-decyl-N,Ndimethyl-3-ammonio-1-propanesulfonate) and ASB14 (tetradecanoylamidopropyl-dimethylammonio-propane-sulfonate).
**Use an ampholyte buffer that corresponds to the pH range of the IEF
separation to be performed. For example, ReadyStrip
with ReadyStrip micro-range IPG strips and ReadyStrip 7-10 buffer with
ReadyStrip pH 7-10 IPG strips. Bio-Lyte 3/10 ampholyte can be used with
all other ReadyStrip IPG strip pH ranges.
****
(w/v) solution
™
micro-range buffers
5.2.2.2. Protein Solubilization Buffer (PSB)
PSB and PSB diluent are provided with the ReadyPrep
protein extraction kit (membrane I), the ReadyPrep protein
extraction kit (cytoplasmic/nuclear), and the ReadyPrep
protein extraction kit (signal) to solubilize the protein
sample. PSB is a proprietary, strongly chaotropic 2-D
rehydration/sample buffer that will solubilize both
hydrophilic and hydrophobic proteins. To make 2 ml of
10
complete 2-D rehydration/sample buffer, add 1.1 ml of PSB
diluent to each 1 g of PSB powder.
Note: Before weighing out the PSB powder, shake the bottle
vigorously for 10–15 sec to break up any clumps and to ensure a
uniform blend of the different components.
Mix the solution until the powder is completely dissolved
(the tube can be warmed to speed dissolution of the solids,
but do not allow the temperature to exceed 30°C). Add
Bio-Lyte ampholyte and Bromophenol Blue as directed in
Section 5.2.2.1 to complete the preparation of the buffer.
Do not add reducing agent.
11
Section 6
References
Görg A et al., The current state of two-dimensional
electrophoresis with immobilized pH gradients, Electrophoresis
21, 1037–1053 (2000)
Hoving S et al., Preparative two-dimensional gel electrophoresis
at alkaline pH using narrow range immobilized pH gradients,
Proteomics 2, 127 (2002)
Herbert B et al., Reduction and alkylation of proteins in
preparation of two-dimensional map analysis: Why, when and
how? Electrophoresis 22, 2046–2057 (2001)
12
Section 7
Product Information
Catalog # Description
Buffer Components
161-0460 CHAPS, 1 g
161-0731 Urea, 1 kg
161-0716 Tris, 500 g
161-0302 Sodium Dodecyl Sulfate (SDS), 1 kg
163-2094 100X Bio-Lyte 3/10 Ampholyte, 1 ml
163-2093 100X Bio-Lyte 7/10 Ampholyte, 1 ml
163-2091 ReadyPrep Proteomic Grade Water
163-2101 Tributylphosphine (TBP), 200 mM, 0.6 ml
163-2109 Iodoacetamide, 30 g
Sample Preparation Kits
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
163-2100 ReadyPrep Sequential Extraction Kit,
5-15 preps
13
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
Cup Loading Accessories (also see Cup Loading Tray
instruction manual, 4006216, and bulletin 2812)
165-4050 Cup Loading Tray, includes 1 pair
moveable electrodes, 1 pack each of
large and small replacement cups
165-4055 Cup Loading Tray with Forceps
165-4051 Large Replacement Cups, 1 pack
(120 count)
165-4052 Small Replacement Cups, 1 pack
(120 count)
Coomassie is a trademark of Imperial Chemical Industries, PLC.
14
Loading...