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Name: Proteomic analysis of rat cerebellum

Description: To study the biochemistry of the cerebellar cortex by using proteomic methods.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2008-11-15

1.1.2 Responsible person or role

Affiliation: Centre for Cellular and Molecular Biology

(i) Name: Purnima Bhargava

(ii) Postal address: Uppal Road, Tarnaka,
Hyderabad-500 007
India

(iii) Email address: purnima@ccmb.res.in

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Cerebellum
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 7M UREA, Thiourea 2M, CHAPS 4%, 18mM Tris (pH 8.0), Trizma 14mM, Triton X-100 0.2%, DTT 50mM, IPGBuffer 2% and trace amount bromophenol blue.

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 Bio-Sciences AB
Model: Immobiline Dry Strip pH 3-10 NL
Model number: 17-6001-15
Batch number: 10013248

3.2.3 Physical dimensions

X: 128 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

sigmoidal pH 3 - 10

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 32:1

3.2.7 Additional substances in gel

No additional substances

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Cerebellum
  • Volume of sample: 1 mg
  • Loading buffer: 7M UREA, Thiourea 2M, CHAPS 4%, 18mM Tris (pH 8.0), Trizma 14mM, Triton X-100 0.2%, DTT 50mM, IPGBuffer 2% and trace amount bromophenol blue.
  • Volume of loading buffer: 300 µL
  • Volume of mixture loaded in the lane: 250 µL

Loading method: cup loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 100 V, 1.30 h

Hold: 300 V, 2 h

Hold: 500 V, 2 h

Hold: 1000 V, 2 h

Hold: 8000 V, 15.00 h

Hold: 8000 V, 10.00 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

Buffer I- 6M urea, 0.375M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol, 2% DTT
Buffer II- Buffer I with 2.5% iodoacetamide instead of DTT

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 25 min.

Protocol:
25 minutes in buffer I
25 minutes in buffer II

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

Composed of 2 sub-gels (Stacking gel, Running gel).

3.2.2 Gel manufacture

Gel was manufactured using the following reference protocol:

Citation: not provided.
URL: http://www.ncbi.nlm.nih.gov/pubmed/5432063

3.2.3 Physical dimensions

Stacking gel:
X: 140 mm
Y: 10 mm
Z: 1.5 mm

Running gel:
X: 140 mm
Y: 140 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

Stacking gel:
No distribution in a Stacking gel.

Running gel:
logarithmic apparent molecular mass 250 - 18 kDa

3.2.5 Acrylamide concentration

Stacking gel:
5% %

Running gel:
12% %

3.2.6 Acrylamide : Crosslinker ratio

Stacking gel:
Crosslinker: Bisacrylamide
Ratio: 29:1

Running gel:
Crosslinker: Bisacrylamide
Ratio: 29:1

3.2.7 Additional substances in gel

Stacking gel:

No additional substances

Running gel:

No additional substances

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: IPG_transfer.

3.3 Protocol

3.3.1 Buffers

Tris-glycin-SDS (25mM-198mM-0.1%w/v)pH 8.3

3.3.2 Electrophoresis conditions

Running temperature: 25 °C

Hold: 10 mA, 12 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

Coomassie briliant blue R250

5.1.3 Additional reagents and buffers

Solution1: CBB 0.5g, isopropanol 250ml, Glacial acetic acid 100ml, Water 650 ml
Solution2: CBB 0.05g, isopropanol 100ml, Glacial acetic acid 100ml, Water 800 ml
Solution3: CBB 0.02g, Glacial acetic acid 100ml, Water 900 ml
Solution4: Glacial acetic acid 100ml, Water 900 ml

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Protocol:
1.At the end of second dimension gels were washed in MQ for 15 minutes.
2.Soaked in Solution1 and heated for 1.20min and cooled for 30minutes
3.Washed in MQ for 5 minutes.
4.Soaked in Solution2 and heated for 1.20min and cooled for 30minutes
5.Washed in MQ for 5 minutes.
6.Soaked in Solution3 and heated for 1.20min and cooled for 30minutes
7.Washed in MQ for 5 minutes.
8.Soaked in solution 4 and heated for 1.20min and cooled for 30minutes
9.Above is repeated till the proper distaining.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

camera

6.1.2 Name of equipment

Manufacturer: Syngene
Model: Dyversity
Model number: Unknown

6.1.3 Software

Manufacturer: Syngene
Model: GeneSnap
Model number: 6.08

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

According to manufacturer's protocol

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

cereb21rat (format: TIFF)

7.1.2 Dimensions

Width: 1141 px

Height: 1276 px

7.1.3 Resolution

72 ppi

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

cereb21rat.tif

7.1.6 Standard image orientation

Yes

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