Name: Basic proteins of the dlPFC region of the human brain

Description: Basic proteins (pH 6-11) of the dorso lateral prefrontal cortex region of the human brain separated by 2-DE

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2008-04-01

1.1.2 Responsible person or role

Affiliation: UCD

(i) Name: Ciara McManus

(ii) Postal address: UCD Conway Institute of Biomolecular and Biomedical Research,
UCD,
Belfield, Dublin, Ireland

(iii) Email address: ciara.mcmanus@ucd.ie

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Dorso later prefrontal cortex (dlPFC) region of the human brain
    2. Sample type: Control sample
    3. URL: http://www.stanleyresearch.org/dnn/

2.1.2 Loading buffer

  1. 9.5 M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT, Protease Inhibitors
  2. 8 M urea, 0.5% w/v CHAPS, 0.2% w/v DTT, 0.2% w/v Pharmalyte pH 6-11

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

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 1:3

3.2.7 Additional substances in gel

DeStreak rehydration solution (GE Healthcare, 17-6003-19)

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Dorso later prefrontal cortex (dlPFC) region of the human brain
  • Volume of sample: 300 µg
  • Loading buffer: 8 M urea, 0.5% w/v CHAPS, 0.2% w/v DTT, 0.2% w/v Pharmalyte pH 6-11
  • Volume of loading buffer: 350 µL

Loading method: paper bridge loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 200 V, 16 h

Hold: 150 V, 1 h

Hold: 300 V, 1 h

Hold: 600 V, 1 h

Hold: 3500 V, 75 h

Hold: 8000 V, 10 min

Hold: 8000 V, 3 h

Hold: 100 V, 72 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M urea containing 30% v/v glycerol, 2% w/v SDS, 0.05 M Tris-HCl, pH 8.8 and 0.01% w/v Bromophenol blue with the addition of 1% w/v DTT

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 22 °C.

Duration: 15 min.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

6 M urea containing 30% v/v glycerol, 2% w/v SDS, 0.05 M Tris-HCl, pH 8.8 and 0.01% w/v Bromophenol blue with the addition of 4.7% w/v iodoacetamide

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 22 °C.

Duration: 15 min.

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: http://nationaldiagnostics.com/images/ec890_protocol.pdf,http://nationaldiagnostics.com/images/ec890_protocol.pdf, page(s) http://nationaldiagnostics.com/images/ec890_protocol.pdf (2005).
URL: http://nationaldiagnostics.com/images/ec890_protocol.pdf

3.2.3 Physical dimensions

X: 240 mm
Y: 200 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 6 - 150 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: paper bridge loading.

3.3 Protocol

3.3.1 Buffers

25mM Tris, 192mM Glycine and 0.1% SDS.

3.3.2 Electrophoresis conditions

Running temperature: 15 °C

Hold: 150 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

Silver Nitrate

5.1.3 Additional reagents and buffers

Fixer 40% Ethanol, 10% Acetic Acid, ddH2O
Sensitising Sodium Acetate, Sodium Thiosulphate, Ethanol
Milli-Q
Developing Sodium Carbonate, formaldehyde
Stopping - EDTA

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Temperature: 22 °C.

Duration: 5 h.

Detection is described in the following reference protocol:
Citation: Electrophoresis,21, page(s) 3666-3672 (2000).
URL: http://www3.interscience.wiley.com/journal/75505007/abstract

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)

Brain dlPFC 6-11 (format: PNG)

7.1.2 Dimensions

Width: 550 px

Height: 568 px

7.1.3 Resolution

72 dpi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

C:\Documents and Settings\Ciara McManus\My Documents\Bridge loading 6-11 gels (stanley series)\Database\BRAIN_DLPFC_6-11.png

7.1.6 Standard image orientation

Yes

<