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)

    1. Sample name: Pooled Standard
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 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

1

3.2.9 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

1

3.2.9 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 scanner

6.1.2 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 exposure

6.2.2 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.tif

7.1.6 Standard image orientation

Yes

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