Bio-Rad ReadyPrep 2-D Starter Kit User Manual
ReadyPrep
™
2-D Starter Kit
Instruction Manual
Catalog Number
163-2105
For technical service
call your local Bio-Rad office or
in the U.S. call 1-800-4BIORAD
(1-800-424-6723)
On the Web at http://www.discover.bio-rad.com
Table of Contents
Section 1 Introduction ......................................................................1
Section 2 Kit Components............................................................1-2
Section 3 Storage ............................................................................2
Section 4 Instructions for Use ..........................................................3
Section 5 Appendix........................................................................22
Section 6 Product Information........................................................25
Section 1
Introduction
The ReadyPrep™2-D starter kit was designed as a single-use kit to familiarize first-time users with
the utilization of the Bio-Rad PROTEAN
®
IEF cell and ReadyStrip™IPG strips. The kit provides all
the reagents necessary, including a protein control, to successfully perform two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The kit contains sufficient reagents to perform 2-D
PAGE of at least 6 samples with any of the three SDS-PAGE formats Bio-Rad offers. The amount
of sample applied to each gel is sufficient so that a large number of protein spots can be readily
detected when using either Bio-Safe
™
Coomassie* stain or Coomassie Brilliant Blue R-250 stain.
This manual provides instructions on how to perform 2-D PAGE with ReadyStrip IPG strips using
the Bio-Rad PROTEAN IEF cell and the control E. coli protein sample.
Section 2
Kit Components
ReadyPrep E. coli Protein Sample. One vial. Lyophilized. Each vial of reconstituted
ReadyPrep E. coli protein sample contains 2.7 mg of total protein at a concentration of 1.35
mg/ml.
ReadyPrep Rehydration/Sample Buffer. One vial. Lyophilized. Each vial of reconstituted
ReadyPrep rehydration buffer contains 10 ml of 8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT),
0.2% (w/v) Bio-Lyte
®
3/10 ampholytes, and Bromophenol Blue (trace).
Nanopure Water. One bottle containing 15 ml of sterile nanopure water.
Equilibration Buffer I. Two vials, each containing a stirbar. Lyophilized. Each vial of reconstitut-
ed equilibration buffer I contains 20 ml of 6 M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), 20%
glycerol, and 2% (w/v) DTT.
Equilibration Buffer II. Two vials, each containing a stirbar. Lyophilized. Each vial of reconstituted equilibration buffer II contains 20 ml of 6 M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), and
20% glycerol.
30% Glycerol Solution. One bottle containing 70 ml of sterile 30% (v/v) glycerol.
Iodoacetamide. Two vials. Each vial contains 0.5 gr of an ultrapure grade of iodoacetamide.
*Coomassie is a trademark of Imperial Chemical Industries PLC.
1
Overlay Agarose. One bottle containing 50 ml of 0.5% low melting point agarose in 25 mM
Tris, 192 mM glycine, 0.1% SDS, and a trace of Bromophenol Blue.
Instruction Manual. One.
Note:
CHAPS is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a
zwitterionic detergent.
Bio-Lyte
®
3/10 ampholytes is a mixture of carrier ampholytes, pH 3–pH 10.
Other Materials Needed
• PROTEAN IEF cell
• ReadyStrip IPG strips, pH 4-7 (7 cm, 11 cm, or 17 cm)
• IEF focusing tray with lid (same size as IPG strips)
• Electrode wicks, precut
• Blotting filter papers
• Mineral oil
• Forceps
• Pipets for volumes ranging from 4-1000 µl
• Stirplate
• Plastic (Saran) wrap
• 8-16% SDS-PAGE gels (Ready Gel
®
, Criterion, or PROTEAN II Ready Gel precast gels
• SDS-PAGE electrophoresis cell (Mini-PROTEAN
®
3, Ready Gel, Criterion, or PROTEAN II XL cell)
• Power supply appropriate for SDS-PAGE system
• Tris/glycine/SDS running buffer
• SDS-PAGE protein stain (Bio-Safe Coomassie or Coomassie Brilliant Blue R-250)
• IEF protein stain (IEF stain or Bio-Safe Coomassie)
• Destain solution (40% methanol, 10% acetic acid)
• Disposable rehydration/equilibration trays with lid (same size as IPG strips)
• SDS-PAGE gel staining trays
• 100 ml graduated cylinders
• High-purity water
Section 3
Storage
Store the entire kit at 4ºC.
2
Section 4
Instructions for Use
Introduction
This 2-D starter kit allows you to successfully separate a complex protein extract by 2-D PAGE
using IPG strips and the Bio-Rad PROTEAN IEF cell. The buffer formulations as well as the
following protocol have been optimized for the E. coli protein sample included in the kit. Before
beginning, decide which length IPG strips you wish to use and how many you wish to focus. The
kit contains sufficient reagents for the separation of the E. coli protein sample on six 17 cm IPGs,
ten 11 cm IPGs or sixteen 7 cm IPGs. The user has the flexibility to choose among three SDSPAGE formats (7 cm IPG strips and Mini-PROTEAN
®
3 Ready Gel precast gels, 11 cm IPG strips
and Criterion
™
gels or 17 cm IPG strips and PROTEAN®II Ready Gel® precast gels). The mini and
Criterion formats have the advantage that the entire 2-D electrophoresis process can be completed within a 24 hour period. The flow chart in Figure 1 can be used to estimate the amount of time
necessary to complete each of the steps of the 2-D process.
The instructions below describe the use of the E. coli protein sample with six IPG strips. Two of
these strips will be stained after the first dimension to illustrate the successful completion of the
IEF step. The remaining IPGs will be carried through the second-dimension separation. The SDSPAGE gels can be stained with either Bio-Rad’s Bio-Safe Coomassie stain or with Coomassie
Brilliant Blue R-250 stain.
3
The ReadyPrep 2-D starter kit has been optimized for pH 4-7 ReadyStrip IPG strips and either
Bio-Safe Coomassie or Coomassie Brilliant Blue R-250 stain. Strips of pH 5-8 can be used but
the results will not match the enclosed gel images. Strips of pH 3-10 are not recommended
unless the protein sample is further diluted into rehydration/sample buffer at least 4-fold. Other
stains can also be used, but again the protein sample needs to be diluted if a more sensitive
stain is used.
4
Rehydration Setup
30 min
Rehydration Setup
30 min
IPG Rehydration
12 hours
IPG Rehydration
12 hours
IPG Rehydration
12 hours
Isoelectric Focusing
5 hours
Isoelectric Focusing
5.3 hours
Isoelectric Focusing
7 hours
IEF stain Subset of
IPGs (Optional)
60 min
IEF stain Subset of
IPGs (Optional)
60 min
IPG Equilibration
for SDS-PAGE
30 min
IPG Equilibration
for SDS-PAGE
30 min
Store IPGs at
-70°C
Mounting IPGs
30 min
Mounting IPGs
30 min
SDS-PAGE
40 min
Destain IPG Strips
60+ min
SDS-PAGE
65 min
Bio-Safe stain
Water Wash
20 min
Rehydration Setup
30 min
Staining
60 min
Staining
60 min
Mounting IPGs
30 min
SDS-PAGE
5.5 hours
Equilibration
30 min
Destain IPG Strips
60+ min
Bio-Safe stain
Water Wash
20 min
Destaining
30-120 min
Destaining
30-120 min
Staining
60 min
Bio-Safe stain
Water Wash
20 min
Destaining
30-120 min
7 cm 11 cm 17 cm
Day 1
Day 2
Day 3
Fig. 1. Flow Chart for 2-D SDS-PAGE.
4.1 Sample Preparation
1 Remove the bottle of rehydration/sample buffer, the bottle of nanopure water and the vial
of E. coli protein sample from the kit. All 3 bottles have a GREEN cap.
2 Remove the desired number of pH 4-7 ReadyStrip IPG strips from the -20ºC freezer and
set aside. (Six strips are recommended: 2 to stain after IEF, 4 to be used for seconddimension SDS-PAGE).
3 Place one disposable rehydration/equilibration tray of the same size as the IPG strips to
be run onto the bench with the sloped end facing to the right (See Figure 2).
4 Remove the crimp and stopper from the bottle of rehydration/sample buffer and reconsti-
tute the lyophilized powder by adding 6.1 ml of the nanopure water supplied with the kit.
Swirl the bottle gently at intervals until all the solids are completely dissolved. The bottle will
chill as the urea in the solids dissolves. The bottle can be warmed slightly in the palm of the
hand to expedite this buffer reconstitution process. Take care not to warm the solution above
30ºC.
5 Open the vial of E. coli protein sample and reconstitute the lyophilized protein by adding
2 ml of the freshly prepared rehydration/sample buffer. Replace the rubber stopper and
gently invert the vial 4 to 6 times to completely reconstitute the E. coli proteins. Do NOT place
the vial on ice as the urea will crystallize out of the solution.
4.2 Sample Application During Rehydration
The amount of reconstituted E. coli protein sample to load per IPG strip is indicated in
Table 1. These quantities are sufficient to easily visualize a large array of protein spots
when the second-dimension SDS-PAGE gel is stained with Bio-Safe Coomassie stain or
Coomassie Brilliant Blue R-250 stain.
1. Using Table 1 below, pipet the indicated volume of the reconstituted E. coli protein sample
as a line along the back edge of channel #1. The line of sample should extend along the
whole length of the channel except for about 1 cm at each end. Take care not to introduce any bubbles which may interfere with the even distribution of sample in the strip
(See Figure 2. Sample loading of rehydration/equilibration trays).
Strip Length 7 cm 11 cm 17 cm
Sample Volume 125 µl 185 µl 300 µl
Protein Loaded 169 µg 250 µg 405 µg
Table 1.
The increased sensitivity of the Bio-Safe Coomassie stain over the R-250 stain allows you to
dilute the stock of E. coli protein sample up to 4-fold into rehydration/sample buffer and still
achieve excellent images. If you choose to stain the SDS-PAGE gels with silver stain (such as
Bio-Rad’s Silver Stain Plus stain, catalog # 161-0449) or a fluorescent stain (such as
SYPRO* Ruby Stain, catalog # 170-3125) use a 4-fold diluted E. coli protein sample (diluted into
rehydration/sample buffer) and the rehydration volumes per strip indicated below.
*SYPRO is a trademark of Molecular Probes, Inc.
5
2. Repeat this process for the remaining samples by pipetting the indicated volume of
sample into adjacent channels. It is best to place samples on both sides of the tray as in
Figure 2. Maintaining the same numbering system throughout the 2-D PAGE process
helps to avoid confusing the samples. Also, during the IEF step when strips are placed on
both sides of the focusing tray, the weight of the tray lid is evenly distributed over all the
samples so contact between the IPGs and the electrode remains uniform.
3. When all the protein samples have been loaded into the rehydration/equilibration tray as
pictured in Figure 2, using forceps, peel the coversheet from one of the pH 4-7
ReadyStrip IPG strips, as in Figure 3.
6
Fig. 2. Sample loading of rehydration/equilibration trays. Pipet the sample along the back edge of
the tray channel except for about 1 cm at each end. Note the even distribution of the sample along the edge
of the channel. The figure shows the last of six samples being pipeted in the tray.
Fig. 3. Removing the cover sheet from the ReadyStrip IPG strip.
Gently place the strip gel side down onto the sample as illustrated in Figure 4. The “+”
and “pH 4-7” should be legible and positioned at the left side of the tray. Take care not to
get the sample onto the plastic backing of the strips as this portion of the sample will not
be absorbed by the gel material. Also take care not to trap air bubbles beneath the strip.
If this happens, carefully use the forceps to lift the strip up and down from one end until
the air bubbles move to the end and out from under the strip.
7
Fig. 4. Placing IPG strips gel side down onto the protein samples.
Rehydration of the IPG strips is a critical
step in the 2-D process. The method
shown in Figures 2-4 allows for consis-
tently even distribution of the sample
along the length of the strip. If the sample
appears unevenly distributed or areas of
the strip were not wetted with sample,
hold the strip at one end with the forceps
and slide the strip back and forth several
times along the length of the channel.
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