MIAPE document  
Gel Electrophoresis MIAPE document  
Based on the original guidelines: MIAPE GE v1.4  
 
1. General features
ID #:
1082
Date stamp:
10/3/2008
Name:
DIGE study of effects of treatment after 24 hrs
Last update:
2/9/2011 12:01:56 PM
Version:
1
 
Electrophoresis type:
difference gel electrophoresis
 
2. Sample (1/2)
ID #:
1467
Name:
2D DIGE labelling.
Description:
Labelled with Cy2, Cy3 and Cy5 fluorescent labels.

Control1 Cy3 - Treatment1 Cy5 - IntStand1 Cy2
Control2 Cy3 - Treatment2 Cy5 - IntStand2 Cy2
Control3 Cy5 - Treatment3 Cy3 - IntStand3 Cy2
Control4 Cy5 - Treatment4 Cy3 - IntStand4 Cy2

Each gels samples were mixed together before being combined with rehydration buffer
 
2. Sample (2/2)
ID #:
1468
Name:
Picking Gel samples
Description:
400 g of a pooled sample made from all biological replicates of treated and untreated samples.
 
3. Protocol (1/2)
Name:
2D pick gel protocol
 
3.1 First dimension (1/2)
Name:
First dimention IEF protocol
Ordinal number for this dimension:
1
Separation method employed:
isoelectric focusing (IEF)
 
3.1.1 Loading buffer (1/2)
Name:
Rehydration buffer
Type:
rehydration buffer
Comments:
460l of rehydration buffer (8M urea, CHAPS and Bromophenol blue) was added to the protein pellet.
 
3.1.1.1 Buffer components
chemical substance - Urea - 12(g)
chemical substance - CHAPS - 2(%)
chemical substance - Bromothymol Blue - 0.001(g)
chemical substance - Dithiothreitol (DTT) - 3.4(mg/ml)
chemical substance - Ampholyte Buffer - 5.5(microliter per milliliter)
 
3.1.1 Loading buffer (2/2)
Name:
Sample Buffer
Type:
sample buffer
Comments:
Equal volumes of 2 x sample buffer was added to samples.
 
3.1.1.1 Buffer components
chemical substance - Urea - 7(M)
chemical substance - Thiourea - 2(M)
chemical substance - CHAPS - 2(weight-volume percentage)
chemical substance - Dithiothreitol (DTT) - 2(weight-volume percentage)
chemical substance - Ampholyte Buffer - 2(%)
 
3.1.2 Gel matrix
ID #:
1320
Name:
immobiline dry strip
Gel manufacture:
GE Healthcare
Physical dimensions:
x: 240
y: 3
z: 0.5
Unit: millimeter
PH range and distribution:
Lower limit: 3
Upper limit: 10
Range type: linear distribution
Acrylamide concentration:
4
Acrylamide : Bisacrylamide ratio:
4 : 3
 
3.1.2.4 Sample application
Name:
sample loading
Comments:
450 l of the rehydration buffer and sample mix was loaded into one lane of the immobiline DryStrip reswelling tray (GE healthcare).
 
3.1.3 Electrophoresis Protocol
Name:
focusing protocol
Electrophoresis conditions:
Step Voltage Time Voltage Mode
1 300 V 3 h Step
2 600 V 3 h Gradient
3 1000 V 3 h Gradient
4 8000 V 3 h Gradient
5 8000 V 4 h Step
 
3.1 Second dimension (2/2)
Name:
Second dimension
Ordinal number for this dimension:
2
Separation method employed:
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),
 
3.1.2 Gel matrix
ID #:
1321
Name:
polyacrylamide gel
Gel manufacture:
30 % Arcylamide 188 ml
1.5 M Tris-Cl pH 8.8 113 ml
dd-H20 140 ml
10% SDS 4.5 ml
10% APS 4.5 ml Add fresh
10% TEMED 0.62 ml

Physical dimensions:
x: 27
y: 21
z: 0
Unit: centimeter
MW range and distribution:
Lower limit: 12000
Upper limit: 120000
Unit: relative molecular mass
Acrylamide concentration:
12.5
 
3.1.2.4 Sample application
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.3 Electrophoresis Protocol
Name:
Electrophoresis protocol
Electrophoresis conditions:
The system was set to run at 5W per gel for 30 min and then 4 hr at 17W per gel.
 
3.1.3.1 Running Buffer
Name:
Running buffer
Type:
electrophoresis buffer
Comments:
Reagent Amount Additional information
Tris base 30.3 g
Glycine 144 g
SDS 10 g
DD-H20 Bring to total of 1000 ml
 
3.2 Inter-dimension Step
Step name:
Equilibration
Protocol:
The strip was equilibrated in 10 ml SDS-equilibration buffer (2% SDS, 50mM Tris-HCL pH 8.8, 6M urea, 30% (v/v) glycerol and 0.002% (w/v) bromophenol blue) plus 100 g DTT for 15 min with shaking. The strip was then incubated for 10 ml SDS-equilibration buffer containing 250 g idoacetamide (IAA) for 15 min with shaking.
 
3. Protocol (2/2)
Name:
2D DIGE protocol
 
3.1 First dimension (1/2)
Name:
First dimention IEF protocol
Ordinal number for this dimension:
1
Separation method employed:
isoelectric focusing (IEF)
 
3.1.1 Loading buffer (1/2)
Name:
Rehydration buffer
Type:
rehydration buffer
Comments:
460l of rehydration buffer was added to the protein pellet.
 
3.1.1.1 Buffer components
chemical substance - Urea - 12(g)
chemical substance - CHAPS - 2(%)
chemical substance - Bromothymol Blue - 0.001(g)
chemical substance - Dithiothreitol (DTT) - 3.4(mg/ml)
chemical substance - Ampholyte Buffer - 5.5(microliter per milliliter)
 
3.1.1 Loading buffer (2/2)
Name:
Sample Buffer
Type:
sample buffer
Comments:
Equal volumes of 2 x sample buffer was added to samples.
 
3.1.1.1 Buffer components
chemical substance - Urea - 7(M)
chemical substance - Thiourea - 2(M)
chemical substance - CHAPS - 2(weight-volume percentage)
chemical substance - Dithiothreitol (DTT) - 2(weight-volume percentage)
chemical substance - Ampholyte Buffer - 2(%)
 
3.1.2 Gel matrix
ID #:
1322
Name:
immobiline dry strip
Gel manufacture:
GE Healthcare
Physical dimensions:
x: 240
y: 3
z: 0.5
Unit: millimeter
PH range and distribution:
Lower limit: 3
Upper limit: 10
Range type: linear distribution
Acrylamide concentration:
4
Acrylamide : Bisacrylamide ratio:
4 : 3
 
3.1.2.4 Sample application (1/2)
Name:
sample loading
Comments:
450 l of the rehydration buffer and sample mix was loaded into one lane of the immobiline DryStrip reswelling tray (GE healthcare).
 
3.1.2.4 Sample application (2/2)
Name:
sample loading
Comments:
450 l of the rehydration buffer and sample mix was loaded into one lane of the immobiline DryStrip reswelling tray (GE healthcare).
 
3.1.3 Electrophoresis Protocol
Name:
focusing protocol
Electrophoresis conditions:
Step Voltage Time Voltage Mode
1 300 V 3 h Step
2 600 V 3 h Gradient
3 1000 V 3 h Gradient
4 8000 V 3 h Gradient
5 8000 V 4 h Step
 
3.1 Second dimension (2/2)
Name:
Second dimension
Ordinal number for this dimension:
2
Separation method employed:
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
 
3.1.2 Gel matrix (1/4)
ID #:
1323
Name:
polyacrylamide gel
Gel manufacture:
30 % Arcylamide 188 ml
1.5 M Tris-Cl pH 8.8 113 ml
dd-H20 140 ml
10% SDS 4.5 ml
10% TEMED 0.62 ml

APS added once solution has been refrigerated for 1 hour:
10% APS 4.5 ml Made up fresh

Physical dimensions:
x: 27
y: 21
z: 0
Unit: centimeter
MW range and distribution:
Lower limit: 12000
Upper limit: 120000
Unit: r
Acrylamide concentration:
12.5
 
3.1.2.4 Sample application
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.2 Gel matrix (2/4)
ID #:
1324
Name:
polyacrylamide gel
Gel manufacture:
30 % Arcylamide 188 ml
1.5 M Tris-Cl pH 8.8 113 ml
dd-H20 140 ml
10% SDS 4.5 ml
10% TEMED 0.62 ml

APS added once solution has been refrigerated for 1 hour:
10% APS 4.5 ml Made up fresh

Physical dimensions:
x: 27
y: 21
z: 0
Unit: centimeter
MW range and distribution:
Lower limit: 12000
Upper limit: 120000
Unit: r
Acrylamide concentration:
12.5
 
3.1.2.4 Sample application (1/2)
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.2.4 Sample application (2/2)
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.2 Gel matrix (3/4)
ID #:
1325
Name:
polyacrylamide gel
Gel manufacture:
30 % Arcylamide 188 ml
1.5 M Tris-Cl pH 8.8 113 ml
dd-H20 140 ml
10% SDS 4.5 ml
10% TEMED 0.62 ml

APS added once solution has been refrigerated for 1 hour:
10% APS 4.5 ml Made up fresh

Physical dimensions:
x: 27
y: 21
z: 0
Unit: centimeter
MW range and distribution:
Lower limit: 12000
Upper limit: 120000
Unit: r
Acrylamide concentration:
12.5
 
3.1.2.4 Sample application (1/2)
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.2.4 Sample application (2/2)
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.2 Gel matrix (4/4)
ID #:
1326
Name:
polyacrylamide gel
Gel manufacture:
30 % Arcylamide 188 ml
1.5 M Tris-Cl pH 8.8 113 ml
dd-H20 140 ml
10% SDS 4.5 ml
10% TEMED 0.62 ml

APS added once solution has been refrigerated for 1 hour:
10% APS 4.5 ml Made up fresh

Physical dimensions:
x: 27
y: 21
z: 0
Unit: centimeter
MW range and distribution:
Lower limit: 12000
Upper limit: 120000
Unit: r
Acrylamide concentration:
12.5
 
3.1.2.4 Sample application (1/2)
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.2.4 Sample application (2/2)
Name:
Sample application
Comments:
Once the gel was set the IPG strip was slotted on to the top of the gel with the acidic end to the left. It was covered with 0.5 % agarose sealing solution, before being assembled into the Ettan DALTsix System.
 
3.1.3 Electrophoresis Protocol
Name:
Electrophoresis protocol
Electrophoresis conditions:
The system was set to run at 5W per gel for 30 min and then 4 hr at 17W per gel.
 
3.1.3.1 Running Buffer
Name:
Running buffer
Type:
electrophoresis buffer
Comments:
Reagent Amount Additional information
Tris base 30.3 g
Glycine 144 g
SDS 10 g
DD-H20 Bring to total of 1000 ml
 
3.2 Inter-dimension Step
Step name:
Equilibration
Protocol:
The strip was equilibrated in 10 ml SDS-equilibration buffer (2% SDS, 50mM Tris-HCL pH 8.8, 6M urea, 30% (v/v) glycerol and 0.002% (w/v) bromophenol blue) plus 100 g DTT for 15 min with shaking. The strip was then incubated for 10 ml SDS-equilibration buffer containing 250 g idoacetamide (IAA) for 15 min with shaking.
 
4. Direct detection
Name of direct detection process:
Staining
Protocol:
To visualize the proteins a SYPRO Ruby protein gel stain (Invitrogen) was used. The gel was fixed in 40% (v/v) MeOH, 10% (v/v) acetic acid for one hour then washed twice with ddH2O for 10 min before staining with SYPRO Ruby gel stain overnight. The gel was then washed in 10% (v/v) MeOH, 7% (v/v) acetic acid for one hour followed by two 5 min washes in ddH2O.
 
4.1 Direct detection agents
SYPRO Ruby - Sufficient to cover gel
 
6. Image Acquisition (1/5)
Name:
Pick gel scan
Protocol:
Dye Excitation Filter (nm) Emission Filter (nm)
Sypro 540/25 595/25
Reference to gel matrix:
1321
 
6.1 Equipment
Name:
Scanning
Type of equipment:
scanner
Calibration:
NO
Model:
EttanTM DIGE Imager
Manufacturer:
Thermo Scientific instrument model
 
6. Image Acquisition (2/5)
Name:
DIGE gel scan
Protocol:
Dye Excitation Filter (nm) Emission Filter (nm)
Cy2 480/30 530/40
Cy3 540/25 595/25
Cy5 635/30 680/30
Reference to gel matrix:
1323
 
6.1 Equipment
Name:
Scanning
Type of equipment:
scanner
Calibration:
NO
Equipment specific parameters:
Default parameter settings
Model:
EttanTM DIGE Imager
Manufacturer:
Thermo Scientific instrument model
Reference (URI):
http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~scanners~ettan_dige_imager?OpenDocument&parentid=63005642&moduleid=166483
 
6.2 Image Acquisition Software
Name:
image acquistion software
Version:
Ettan DIGE Imager software 1.0
Vendor: 
GE Healthcare Life Sciences
 
6. Image Acquisition (3/5)
Name:
DIGE gel scan
Protocol:
Dye Excitation Filter (nm) Emission Filter (nm)
Cy2 480/30 530/40
Cy3 540/25 595/25
Cy5 635/30 680/30
Reference to gel matrix:
1324
 
6.1 Equipment
Name:
Scanning
Type of equipment:
scanner
Calibration:
NO
Equipment specific parameters:
Default parameter settings
Model:
EttanTM DIGE Imager
Manufacturer:
Thermo Scientific instrument model
Reference (URI):
http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~scanners~ettan_dige_imager?OpenDocument&parentid=63005642&moduleid=166483
 
6.2 Image Acquisition Software
Name:
image acquistion software
Vendor: 
GE Healthcare Life Sciences
 
6. Image Acquisition (4/5)
Name:
DIGE gel scan
Protocol:
Dye Excitation Filter (nm) Emission Filter (nm)
Cy2 480/30 530/40
Cy3 540/25 595/25
Cy5 635/30 680/30
Reference to gel matrix:
1325
 
6.1 Equipment
Name:
Scanning
Type of equipment:
scanner
Calibration:
NO
Equipment specific parameters:
Default parameter settings
Model:
EttanTM DIGE Imager
Manufacturer:
Thermo Scientific instrument model
Reference (URI):
http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~scanners~ettan_dige_imager?OpenDocument&parentid=63005642&moduleid=166483
 
6.2 Image Acquisition Software
Name:
image acquistion software
Vendor: 
GE Healthcare Life Sciences
 
6. Image Acquisition (5/5)
Name:
DIGE gel scan
Protocol:
Dye Excitation Filter (nm) Emission Filter (nm)
Cy2 480/30 530/40
Cy3 540/25 595/25
Cy5 635/30 680/30
Reference to gel matrix:
1326
 
6.1 Equipment
Name:
Scanning
Type of equipment:
scanner
Calibration:
NO
Equipment specific parameters:
Default parameter settings
Model:
EttanTM DIGE Imager
Manufacturer:
Thermo Scientific instrument model
Reference (URI):
http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~scanners~ettan_dige_imager?OpenDocument&parentid=63005642&moduleid=166483
 
6.2 Image Acquisition Software
Name:
image acquistion software
Vendor: 
GE Healthcare Life Sciences
 
7. Image (1/13)
Name of indirect detection process:
Gel1-Cy2
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61713_b_STANDARD_CY2.gel
Reference to gel matrix:
1323
 
7. Image (2/13)
Name of indirect detection process:
Gel1-Cy3
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61713_b_CY3.gel
Reference to gel matrix:
1323
 
7. Image (3/13)
Name of indirect detection process:
Gel1-Cy5
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61713_b_CY5.gel
Reference to gel matrix:
1323
 
7. Image (4/13)
Name of indirect detection process:
Gel2-Cy2
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61714_STANDARD_CY2.gel
Reference to gel matrix:
1324
 
7. Image (5/13)
Name of indirect detection process:
Gel2-Cy3
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61714_CY3.gel
Reference to gel matrix:
1324
 
7. Image (6/13)
Name of indirect detection process:
Gel2-Cy5
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61714_CY5.gel
Reference to gel matrix:
1324
 
7. Image (7/13)
Name of indirect detection process:
Gel3-Cy2
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61715_STANDARD_CY2.gel
Reference to gel matrix:
1325
 
7. Image (8/13)
Name of indirect detection process:
Gel3-Cy3
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61715_CY3.gel
Reference to gel matrix:
1325
 
7. Image (9/13)
Name of indirect detection process:
Gel3-Cy5
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61715_CY5.gel
Reference to gel matrix:
1325
 
7. Image (10/13)
Name of indirect detection process:
Gel4-Cy2
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61716_STANDARD_CY2.gel
Reference to gel matrix:
1326
 
7. Image (11/13)
Name of indirect detection process:
Gel4-Cy3
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61716_CY3.gel
Reference to gel matrix:
1326
 
7. Image (12/13)
Name of indirect detection process:
Gel4-Cy5
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/61716_CY5.gel
Reference to gel matrix:
1326
 
7. Image (13/13)
Name of indirect detection process:
Pickgel
Format:
Tagged Image File Format
Dimensions:
X: 2537 pixels
Y: 2041 pixels
Resolution:
252 dots per inch
Bit-depth:
16 bits
Stored in:
http://estrellapolar.cnb.csic.es/proteored/MIAPE/Ficheros/462/PG2.jpg
Reference to gel matrix:
1321