Name: HCR_LCR_Cardiac Map
Description: Large format 2DE separation of rat cardiac proteins
Version: MIAPE: Gel Electrophoresis 1.4
1. General features
1.1.1 Date Stamp
2009-02-18
1.1.2 Responsible person or role
Affiliation: Liverpool John Moores University
(i) Name: Jatin Burniston
(ii) Postal address: Research Institute for Sport and Exercise Sciences
Liverpool John Moores University
Tom Reilly Building
Byrom Street
Liverpool
L3 2ET
United Kingdom
(iii) Email address: j.burniston@ljmu.ac.uk
1.1.3 Electrophoresis type
Two-Dimensional electrophoresis
2. Sample
2.1.1 Sample Name(s)
-
- Sample name: Pooled Standard
- Sample type: Standard
2.1.2 Loading buffer
- 7 mol urea, 2 mol thiourea, 2 % (w/v) CHAPS, 20 mmol DTT and 0.5% (v/v) ampholytes
3. Gel matrix and electrophoresis protocol
3.1 Dimension details
3.1.1 Ordinal number for this dimension
First
3.1.2 Separation method employed
Isoelectric focusing (IEF)
3.2 Gel Matrix
3.2.1 Description of gel matrix
IPG strip
Denaturing
3.2.2 Gel manufacture
Gel was purchased precast.
Manufacturer: GE HEalthcare
Model: 24 cm pH 3-11NL IPG strip
Model number: 17-6003-77
Batch number: unknown
3.2.3 Physical dimensions
X: 24 cm
Y: 3 cm
Z: 0.5 cm
3.2.4 Physiochemical property range and distribution
Non-linear pH 3 - 11
3.2.5 Acrylamide concentration
4 %
3.2.6 Acrylamide : Crosslinker ratio
Crosslinker: Bisacrylamide
Ratio: 4:3
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Lane 1
- Sample: Pooled Standard
- Volume of sample: 1000 µg
- Loading buffer: 7 mol urea, 2 mol thiourea, 2 % (w/v) CHAPS, 20 mmol DTT and 0.5% (v/v) ampholytes
- Volume of loading buffer: 450 µL
Loading method: rehydration loading.
3.3 Protocol
3.3.1 Buffers
No buffer.
3.3.2 Electrophoresis conditions
Running temperature: 20 °C
Hold: 300 V, 3 h
Hold: 600 V, 3 h
Hold: 1000 V, 3 h
Gradient: 1000-8000 V, 3 h
Hold: 8000 V, 4 h
4. Inter-dimension Process
4.1 Protocol
4.1.1 Step name
reduction
4.1.2 Inter dimension buffer
50 mmol Tris-HCl pH 8.8, containing 6 mol urea, 30 % v/v glycerol, 2 % w/v SDS and a trace of bromophenol blue
4.1.3 Additional reagents
10 mg/ml dithiothreitol
4.1.4 Equipment
4.1.5 Protocol
Temperature: 22 °C.
Duration: 15 min.
Protocol:
Strip equilibration
4.1.1 Step name
alkylation
4.1.2 Inter dimension buffer
50 mmol Tris-HCl pH 8.8, containing 6 mol urea, 30 % v/v glycerol, 2 % w/v SDS and a trace of bromophenol blue
4.1.3 Additional reagents
25 mg/ml iodoacetamide
4.1.4 Equipment
4.1.5 Protocol
Temperature: 22 °C.
Duration: 15 min.
Protocol:
Strip equilibration
3. Gel matrix and electrophoresis protocol
3.1 Dimension details
3.1.1 Ordinal number for this dimension
Second
3.1.2 Separation method employed
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
3.2 Gel Matrix
3.2.1 Description of gel matrix
slab gel
Denaturing
3.2.2 Gel manufacture
Gel was manufactured using the following receipe:
Prepare gel mix: for 12.5 % Acrylamide (37.5:1 acrylamide: bis-acrylamide) mix (6 large format gels):
30 % Acrylamide: bis-acrylamide 333 ml
4x Buffer (1.5 M Tris pH 8.8) 250 ml
10 % SDS 8.0 ml
10 % TEMED 1.2 ml
*Cool in fridge for 1 h*
10 % (0.1 g/ ml) Ammonium persulphate 8.0 ml
3.2.3 Physical dimensions
X: 240 mm
Y: 260 mm
Z: 1 mm
3.2.4 Physiochemical property range and distribution
linear apparent molecular mass 10 - 120 kDa
3.2.5 Acrylamide concentration
12.5 %
3.2.6 Acrylamide : Crosslinker ratio
Crosslinker: Bisacrylamide
Ratio: 37.5:1
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Loading method: well loading.
3.3 Protocol
3.3.1 Buffers
10x Running buffer:
75.5 g Tris base
360.5 g Glycine
25 g SDS
ddH2O make up to 2.5 l
Dilute 500 ml of 10x Running buffer with 4500 ml ddH2O to make 5 l of 1x Running buffer for lower tank.
Dilute 400 ml of 10x Running buffer with 1600 ml ddH2O to make 2 l of 2x Running buffer for upper reservoir
3.3.2 Electrophoresis conditions
Running temperature: 20 °C
Hold: 70 mA, 30 min
Hold: 400 mA, 5 h
5. Detection
5.1 Direct detection
5.1.1 Name of direct detection_process
Coomassie blue staining
5.1.2 Direct detection agents
Colloidal Coomassie blue
5.1.3 Additional reagents and buffers
No additional reagents or buffer
5.1.4 Equipment
No specialised equipment.
5.1.5 Direct detection protocol
Temperature: 22 °C.
Duration: 1 h.
Protocol:
CCB Staining
Gel washed 3x 5 minutes in distilled water.
Incubate in colloidal Coomassie blue (Bio-safe; Bio-Rad Laboratories) at room temperature for 1 h.
De-stained in distilled water overnight at room temperature.
6. Image Acquisition
6.1 Acquisition Equipment
6.1.1 Type of equipment
Translumination scanner6.1.2 Name of equipment
Manufacturer: Espon
Model: Epson 1680
Model number: 1680
6.1.3 Software
Manufacturer: Epson
Model: Epson
Model number: 1680
6.1.4 Calibration
Yes (automatic)
6.1.5 Equipment specific parameters
Default (vendor) parameters.6.2 Acquisition Protocol
6.2.1 Image acquisition process
Default exposure6.2.2 Reference to gel matrix
There is only one gel in this document.
7. Image
7.1.1 Image name (or id)
HCR LCR cardiac map (format: TIFF)
7.1.2 Dimensions
Width: 1368 px
Height: 1050 px
7.1.3 Resolution
100 dpi
7.1.4 Bit-depth
16-bit (HighColor)7.1.5 Image location
HCR LCR Cardiac Map.tif7.1.6 Standard image orientation
Yes