Name: 2-DE proteome map of the basic human heart proteins

Description: This map described the pH 6-11 region of the human heart proteome.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2010-02-01

1.1.2 Responsible person or role

Affiliation: University College Dublin

(i) Name: Julie Polden

(ii) Postal address: Conway Institute of Biomolecular and Biomedical Research,
University College Dublin,
Belfied,
Dublin,
Ireland

(iii) Email address: julie.polden@ucd.ie

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Left ventricular tissue lysate from human heart
    2. Sample type: Control sample

2.1.2 Loading buffer

  1. Lysis Buffer: 9.5M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT, protease inhibitor cocktail (Complete Mini). Rehydration Buffer: 8M urea, 0.5% CHAPS, 0.2% DTT, 0.2% Pharmaltye pH 6-11, 1.2% Destreak Reagent (GE Healthcare).

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: Immobiline DryStrip pH 6-9, 18 cm
Model number: 17-6001-97
Batch number: 10008430

3.2.3 Physical dimensions

X: 180 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

linear pH 6 - 11

3.2.5 Acrylamide concentration

0 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 3:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Left ventricular tissue lysate from human heart
  • Volume of sample: 300 µg
  • Loading buffer: Lysis Buffer: 9.5M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT, protease inhibitor cocktail (Complete Mini). Rehydration Buffer: 8M urea, 0.5% CHAPS, 0.2% DTT, 0.2% Pharmaltye pH 6-11, 1.2% Destreak Reagent (GE Healthcare).
  • Volume of loading buffer: 100 µL

Loading method: paper bridge loading.
Additional comment: The IPG strip was passively rehydrated overnight in 350 microlitres rehydration buffer containing 1.2% DeStreak Reagent (GE Healthcare). A sample loading cup was used to ensure the paper bridge remained in contact with the IPG strip.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 150 V, 1 h

Hold: 300 V, 1 h

Hold: 600 V, 1 h

Hold: 3500 V, 16 h

Gradient: 3500-8000 V, 10 min

Hold: 8000 V, 3 h

Hold: 100 V, 1 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M urea, 50 mM TrisCl pH 8.8, 30% glycerol, 2% SDS

4.1.3 Additional reagents

1% DTT

4.1.4 Equipment

4.1.5 Protocol

Temperature: 22 °C.

Duration: 15 min.

Protocol:
Strips immersed in individual wells of a rehydration tray and kept shaking for 15 minutes.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

6 M urea, 50 mM TrisCl pH 8.8, 30% glycerol, 2% SDS

4.1.3 Additional reagents

4.7% Iodoacetamide

4.1.4 Equipment

4.1.5 Protocol

Temperature: 22 °C.

Duration: 15 min.

Protocol:
Strips immersed in individual wells of a rehydration tray and kept shaking for 15 minutes.

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 reference protocol:

Citation: not provided.
URL: http://nationaldiagnostics.com/images/ec890_protocol.pdf

3.2.3 Physical dimensions

X: 250 mm
Y: 200 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 100 - 6 kDa

3.2.5 Acrylamide concentration

12% %

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: IPG strip.
Additional comment: IPG strip was placed on top of the slab gel and held in place with Agarose Sealing Solution (0.5% agarose in SDS Electrophoresis Buffer).

3.3 Protocol

3.3.1 Buffers

25mM Tris, 192mM Glycine, 0.1% SDS

3.3.2 Electrophoresis conditions

Running temperature: 15 °C

Hold: 15 mA, 16 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Silver staining

5.1.2 Direct detection agents

http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/FDFA19E77539CA76C1257628001CF9E6/$file/71717700AL.pdf

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: 3 h.

Detection is described in the following reference protocol:
Citation: Electrophoresis,21, page(s) 3666-3672 (2000).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

Calibrated densitometer

6.1.2 Name of equipment

Manufacturer: Bio-Rad
Model: GS-800 Calibrated Densitometer
Model number: 170-7980

6.1.3 Software

Manufacturer: Bio-Rad
Model: PDQuest
Model number: 7.3.1

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

Filter = Green, Light = Transmissive

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

HEART PH6-11 BRIDGE (format: PNG)

7.1.2 Dimensions

Width: 909 px

Height: 860 px

7.1.3 Resolution

72 dpi

7.1.4 Bit-depth

24-bit (TrueColor)

7.1.5 Image location

HEART PH6-11 BRIDGE.png

7.1.6 Standard image orientation

Yes

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