2D gel Protocol
1. Lysis and Protein Extraction from cells
Prepare cell lysates with Trizol extraction by following Kathleen Lyons’s protocol:
AfCS Procedure Protocol PP00000155, Version 1, 05/12/03 (Ref.1).
This preparation method provides total cellular protein samples that are free of
contaminating nucleic acids and free of protease activity. The protein extracts are
used for analysis of proteins by two-dimensional gel electrophoresis.
2. Determining Protein Concentration of cell lysates
Perform a Bradford protein assay (Bio-Rad Protein Assay Instructions come with
the Protein Assay Kit I, Catalogue No. 500-0001 (Ref.2)) to determine protein
concentration.
3. First dimension Separation: Isoelectric Focusing (IEF)
This protocol uses a 13 cm strip as an example
This protocol is based on that found in the handbook prepared by Amersham
Biosciences: 2-D Electrophoresis using immobilized pH gradients Principles
and Methods 80-6429-60 (Ref.3)
1) Decide what size of IPG strip you will use for your experiment and how much of
your sample to use. The following table provides a range for Sample loading onto
a IPG strip.
Table 1: Suitable sample loads for silver and Coomassie staining
Immobiline
Dry Strip
7cm
11cm
13cm
18cm
24cm
pH
4-7
6-11
3-10, 3-10 NL
4-7
6-11
3-10 L
4-7
6-11
3-10, 3-10 NL
4-7
6-11, 6-9, narrow interval
3-10, 3-10 NL
4-7, 3-7
6-9, narrow interval
3-10, 3-10 NL
Suitable Sample Load (ug of protein)
Silver stain Coomassie stain
4-8
8-16
2-6
10-20
20-40
4-8
15-30
30-60
8-15
30-60
60-120
15-30
45-90
80-170
20-40
20-120
40-240
10-60
50-300
100-600
20-120
75-450
150-900
40-240
150-900
300-1500
75-450
200-1300
400-2000
100-600
2) Prepare the strip holder(s) corresponding to the IPG strip length chosen for the
experiment. Handle the ceramic strip holder with care, as they are fragile.
3) Wash strip holder:
Rinse each strip holder with ddH2O followed by adding a few drops of Ettan
IPGphor Strip Holder Cleaning Solution. Use a toothbrush and vigorous
agitation to clean the strip holder. Rinse well with ddH2O. Thoroughly dry the
strip holders with lint-free tissue prior to use.
4) Prepare fresh rehydration solution
For example: thaw 945uL rehydration stock (stock in the –80°C freezer), add
50uL of 1M DTT and 5uL of IPG buffer solution mix well.
5) Prepare the sample and rehydration solution mixture: add appropriate volume of
rehydration solution into the sample to make up the total volume indicated in the
following table
Table 2. Sample and rehydration solution mixture volume per IPG Strip
IPG Strip length (cm)
7
11
13
18
24
Total volume per strip (uL)
125
200
250
340
450
For example: a sample is determined to have 5.7 ug/uL total protein. Only 150ug
of this protein sample will be run on a 13cm pH 4-7 strip. Therefore, 26.3uL of
this sample plus 223.6 uL of rehydration solution will be mixed up to make 250uL
of total volume. This sample will be used for SYPRO ruby staining.
6) Distribute the rehydration-sample solution mixture evenly in the strip holders. Be
sure to record the boat number with the sample name.
7) Remove the protective cover from the IPG strip starting at the positive (acidic,
pointed or arrow) end. Position the IPG strip in the reswelling holder with the gel
side down and the positive end of the strip against the sloped end of the slot.
8) Overlay each IPG strip with DryStrip Cover Fluid to minimize evaporation and
urea crystallization. This amount is variable (eg. 300-400uL for 7cm and 700800uL for 13cm strips).
9) Run your IPG strip on the Ettan IPGphor II.
Align each strip with the strip length markers present on the IPGphors. Put IPG
strip positive on IPGphor positive side. Place the internal cover over the ceramic
holders and close the lid. Turn on the machine. Use the up and down cursors to
select which protocol you need (check the following table for protocols). Use the
up and down cursors to select how many strips you will be running (up to 6?).
Table 3. Guidelines for Ettan IPGphor with rehydration loading/IEF for
Immobiline Dry Strips. Voltage step and hold mode, 50uA/IPG strip, 0.5% IPG
buffer, 20°C for both rehydration and IEF. Rehydration time 12 hours.
Immobiline DryStrip
Length pH range(s)
7cm*
3-10
3-10NL
4-7
11cm
3-10
4-7
13cm* 3-10
3-10NL
4-7
18cm
3-10
3-10NL
4-7
18cm
Narrow
Intervals
24cm
3-10
3-10NL
4-7
3-7
24cm
Narrow
Intervals
Step and
voltage mode
1SH
2SH
3SH
4SH
5SH
6SH
7SH
Total
1SH
2SH
3SH
4SH
Total
1SH
2SH
3SH
4SH
5SH
6SH
7SH
8SH
Total
1SH
2SH
3SH
4SH
Total
1SH
2SH
3SH
4SH
Total
1SH
2SH
3SH
4SH
Total
1SH
2SH
3SH
4SH
Total
Rehydration loading
Step duration
Voltage (V) (h:min)
20
12:00
100
1:00
500
1:00
1000
1:00
2000
1:00
4000
1:00
8000
4:00
21:00
20
12:00
500
1:00
1000
1:00
8000
1:50
15:50
20
12:00
100
2:00
500
1:00
1000
1:00
2000
2:00
4000
2:00
6000
2:00
8000
8:00
30:00
20
12:00
500
1:00
1000
1:00
8000
4:00
18:00
20
12:00
500
1:00
1000
1:00
8000
7:30
21:30
20
12:00
500
1:00
1000
1:00
8000
8:20
22:20
20
12:00
500
1:00
1000
1:00
8000
10:30
24:30
Volt-hours
(kVh)
0.24
0.1
0.5
1
2
4
32
39.84
0.24
0.5
1
12.5
14.24
0.24
0.2
0.5
1
2
8
12
64
87.94
0.24
0.5
1
30.5
32.24
0.24
0.5
1
58.5
60.24
0.24
0.5
1
62.5
64.24
0.24
0.5
1
94.5
96
SH = Step and Hold
*The 7 and 13cm protocol are currently the only protocols tested. All other protocols are
suggested by the manufacturer.
10) Once the program is complete, turn off the power and remove your samples.
11) IPG strips can either be stored in a plastic Petri dish at -80°C (gel side facing up)
or directly following two equilibration steps for the further SDS-PAGE analysis.
12) The ceramic holders are cleaned with a special cleaning solution made by
Amersham (see step 3). The gold plates on the IPGphors are cleaned with water
only.
4. Second dimension Separation: SDS-Page
This protocol uses 140 x 150 x 1mm SDS-page (middle gel) as an example
1) Make a SDS polyacrylamide gel without the upper stack, leaving about 50mm of
room from the top.
2) If your strips are stored at -80°C, remove the IPG strips from the freezer and
allow them to thaw for 10 to 20 minutes prior to equilibration of strips. Always
use tweezers when handling strips. Tweezers should only touch the strips at the
edges
3) Equilibration of IPG strips prior to SDS PAGE:
First Equilibration with reductant: 15 min reduction with SDS-Equilibration
buffer plus dithiothreitol (DTT).
Second Equilibration with alkylating agent: 15 min alkylaion with SDSEquilibration buffer plus iodoacetamide (IAA).
Both procedures are conducted while shaking the samples in solution. Make sure
the procedure takes place while the IPG strips are facing upwards.
4) Prepare a protein standards marker: heat the protein standards at 90°C for 3
minutes before applying it to a 50mm x 50mm piece of filter paper.
For example, in Ref.3 for 255 x 196 x 1 mm gel, apply 200 to 1000 ng of each
protein for Commasssie staining and about 10 to 50 ng of each protein for silver
staining . (In fact 1ug of each protein of unstained protein marker is used to 140 x
150x 1 mm gel by Dr. Litchfield lab for SYPRO ruby staining gel)
5) Wash IPG strips with SDS PAGE running buffer (1x) before loading to the
second-dimension gel in order to get rid of the free DTT and IAA.
6) Apply the equilibrated IPG strips to the second-dimension gel between the two
glass plates. Make sure the IPG plastic backing is against ONE of the glass plates.
Leave room on one side of the strip and place the filter paper with protein marker
here.
Tip: use pipette tips to push the strip down into two plates so it can to make
contact with the gel.
7) Seal the IPG strip in place: Add about 1mL of agarose sealing mixture over the
gel strip and filter paper to completely cover them. Allow 10 to 15 minutes to
solidify.
Tip: If Agarose sealing solution is already made and in a falcon tube, open the
tube and place it face down in a glass beaker before putting the solution in the
microwave. This will make for an easier (and cleaner) melting process
8) Run the gel: Generally, the higher the voltage, the faster your samples will run
through the gel. However, at too high of a voltage melting as well as a pour visual
resolution of your gel will occur.
For Example: for a 12%, 14cm gel (middle-gel) it typically is run at 200V and
0.3A for 4.5hrs.
9) Stain the gel with Coomassie, Silver or SYPRO Ruby staining to determine
protein levels or with ProQ Diamond staining to detect phosphorylated proteins.
Reagents
1% Bromophenol Blue Stock Solution:
Solution Component
Bromophenol blue
Tris-base
Deionized water (Milli Q)
Amount (10 mL)
100 mg
60 mg
Up to 10 mL
Rehydration Buffer:
Solution Component
Amount (10 mL)
Urea
4.2 g
Thiourea
1.52 g
CHAPS
0.4 g
1 M DTT*
500 µL
100% IPG buffer (Ampholines)*
50 µL
1% Bromophenol blue
200 µL
Deionized water (Milli Q)
Up to 10 mL
*If freezing Rehydration Buffer do not add DTT or IPG buffer until ready to use
SDS Equilibration Buffer:
Solution Component
1 M Tris, pH 8.8
Urea
100% Glycerol
10% SDS
1% Bromophenol Blue
Deionized water (Milli Q)
Amount (200 mL)
10 mL
72.07 g
60 mL
40 mL
400 µL
to 200 mL
This is a stock solution. Prior to use DTT or iodoacetamide are added.
For SDS equilibration buffer with DTT-> add 100 mg DTT to 10 mL of buffer
For SDS equilibration buffer with IAA-> add 250 mg IAA to 10 mL of buffer
12% SDS Polyacrylamide Gel:
Solution Component
Deionized water (Milli Q)
30% Acrylamide
1.5 M Tris, pH 8.8
10% SDS
10% APS
TEMED
Amount (10 mL)
3.3 mL
4.0 mL
2.5 mL
100µL
100µL
4µL
For different gel compositions please consult the source: Sambrook, J., and Russell, D.W.,
Molecular Cloning: A laboratory manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York
Agarose Sealing Mixture:
Solution Component
1X TAE Buffer
Agarose
1% Bromophenol Blue
Amount
50 mL
0.5 g
100 µL
Microwave to boiling point to dissolve
SDS PAGE Running Buffer (10x):
Solution Component
Amount (4L)
Glycine
Tris base
SDS
ddH2O
576g
121.1g
40g
top up to 4L