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Name: Control Gel

Description: Protein expression in the Inferior mesenteric vein (IMV) of matched-control patients.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-03-01

1.1.2 Responsible person or role

Affiliation: Researcher

(i) Name: Ian Nordon

(ii) Postal address: St George's Vascular Institute

(iii) Email address: inordon@sgul.ac.uk

1.1.3 Electrophoresis type

Difference gel electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Control Samples
    2. Sample type: Control sample

2.1.2 Loading buffer

  1. Urea (Amersham Biosciences)8M, 48g Pharmalyte (Amersham Biosciences)0.2%, 2.5(ml) CHAPS (VWR International), 0.5%, 0.5g DTT (Sigma), 2%, 0.4g In 100ml double distilled water. Store at -80ºC

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 manufactured using the following reference protocol:

Citation: Industry standrard,n/a, page(s) n/a (2003).
URL: http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000149.pdf?pid=PP00000149

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 3 - 10

3.2.5 Acrylamide concentration

8-12 % (linear)

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 19:1

3.2.7 Additional substances in gel

APS 38ml
Glycerol 149ml
Temed 44ml
Tris-SDS 151ml
Water 686ml

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Control Samples
  • Volume of sample: 50 µL
  • Loading buffer: Urea (Amersham Biosciences)8M, 48g Pharmalyte (Amersham Biosciences)0.2%, 2.5(ml) CHAPS (VWR International), 0.5%, 0.5g DTT (Sigma), 2%, 0.4g In 100ml double distilled water. Store at -80ºC
  • Volume of loading buffer: 320 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: room temperature (no cooling device).

Hold: 500 V, 10 min

Hold: 500 V, 110 min

Hold: 1200 V, 45 min

Hold: 2000 V, 30 min

Hold: 2500 V, 20 min

Hold: 3500 V, 60 min

Hold: 3500 V, 600 min

Hold: 10000 V, 180 min

Hold: 10000 V, 60 min

Hold: 500 V, 60 min

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

Equilibration buffer
Urea Amersham Biosciences 8M 48
SDS Invitrogen 2% 20
Glycerol Invitrogen - 300g
Tris pH 8.8 Invitrogen 50mM 33(ml)

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Protocol:
Incubation with DTT and Equilibration buffer (15/60)
Incubation with Iodoacetamide and Equilibration buffer (15/60)
Rinse in water

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: Industry standrard,n/a, page(s) n/a (2003).
URL: http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000149.pdf?pid=PP00000149

3.2.3 Physical dimensions

X: 260 mm
Y: 210 mm
Z: 2 mm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 200 - 10 kDa

3.2.5 Acrylamide concentration

8-12 % (linear)

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 19:1

3.2.7 Additional substances in gel

APS 38ml
Glycerol 149ml
Temed 44ml
Tris-SDS 151ml
Water 686ml

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: well loading.

3.3 Protocol

3.3.1 Buffers

Tris-Glycine SDS Running Buffer

3.3.2 Electrophoresis conditions

Running temperature: 10 °C

Hold: 600 V, 18.5 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

Cyanine dyes (Cy2/3/5)

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

Manufacturer: GE Healthcare
Model: Typhoon
Model number: 9400

5.1.5 Direct detection protocol

Detection is described in the following reference protocol:
Citation: Other,n/a, page(s) n/a (2003).
URL: http://www.currentprotocols.com/protocol/mb1023

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

fluorescent scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare
Model: Typhoon
Model number: 9400

6.1.3 Software

Manufacturer: GE Healthcare
Model: ImageQuant
Model number: 5.2

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

30 minutes/gel
100 micrometer pixel size
Cy2 500nM
Cy3 545nM
Cy5 550nm

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

Control (format: TIFF)

7.1.2 Dimensions

Width: 2800 px

Height: 2080 px

7.1.3 Resolution

100 µm/px

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

E:\Control1.tif

7.1.6 Standard image orientation

Yes

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