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Name: Analysis of Pig oocyte

Description: The protein production of mammalian embryos is known relatively insufficient. Unlike the genome, the proteome itself is forceful reflecting an environmental signal. Until now the lack of sensitivity has remained an uncertain block for the global introduction of proteomics into the field of mammalian embryology. Therefore, we analyzed protein expression level of pig oocyte.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2010-10-01

1.1.2 Responsible person or role

Affiliation: Professor

(i) Name: Jin, Dong IL

(ii) Postal address: 79 Daehangno, yoseong-gu, Daejeon, 305-764, Chungnam National University, Korea

(iii) Email address: dijin@cnu.ac.kr

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Pig oocyte
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 1% SDS, 1 mM PMSF, 1X protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl(pH 7.0)
  2. 7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT, 1 mM PMSF, protease inhibitor, 40 mM Tris-HCl(pH7.0)

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: pre-cast immobilized dry strips pH3-10 non-linear
Model number: 17-1235-01
Batch number: 10035230

3.2.3 Physical dimensions

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

3.2.4 Physiochemical property range and distribution

non-linear pH 3 - 10

3.2.5 Acrylamide concentration

3-10 % (non-linear)

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 29:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Pig oocyte
  • Volume of sample: 1 mg
  • Loading buffer: 1% SDS, 1 mM PMSF, 1X protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl(pH 7.0)
  • Volume of loading buffer: 100 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

anode rehydration buffer (6 M urea, 2 M thiourea, 4% CHAPS, 0.4% DTT, 2% v/v IPG buffer pH 4-7)
cathode modified buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2.5% DTT, 10% v/v isopropanol, 5% v/v glycerol, 2% v/v IPG buffer pH 6-11)

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 50 V, 1000 min

Hold: 100 V, 100 min

Hold: 300 V, 100 min

Hold: 600 V, 100 min

Hold: 1000 V, 100 min

Hold: 3000 V, 100 min

Hold: 5000 V, 100 min

Hold: 8000 V, 400 min

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

After the first dimensional IEF, IPG gel strip were placed in an equilibration solution (6 M urea, 2% SDS, 50% v/v glycerol, 2.5% acrylamide, 1.875 M Tris-HCl, pH 8.8) containing 5 mM TBP for 20min with gentle shaking.

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: GE Healthcare
Model: Multiphor II IEF system
Model number: GE Healthcare Bio-Sciences, Uppsala, Sweden

Manufacturer: unknown
Model: unknown
Model number: unknown

4.1.5 Protocol

Temperature: 20 °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: Proteomics,5, page(s) 4264-4273 (2005).
URL: not provided.

3.2.3 Physical dimensions

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

3.2.4 Physiochemical property range and distribution

non-linear apparent molecular mass 10 - 200 kDa

3.2.5 Acrylamide concentration

8-16 % (non-linear)

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 29:1

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

*. 5X SDS Running Buffer (20L)
Glycine (amresco, 0167) 1440g
SDS (Lauryl Sulfate, Sigma, L-3771) 100g
Tris base (amresco, 0826) 300g
Water up to 20L

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 30 mA, 1 h

Hold: 70 mA, 24 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 brilliant blue (CBB) G-250

5.1.3 Additional reagents and buffers

Fixer (2L)
MeOH (TEDIA, MS-1922) 800ml (net 40%)
Phosphoric Acid 100ml (net 5%)
Water 1100ml


Coomassie (1L)
Ammonium sulfate 170g (net 17%)
Phosphoric Acid 36ml (net 3%)
Coomassie G-250 (Fluka, 27815) 1g
MeOH (TEDIA, MS-1922) 340ml (net 34%)
Water up to 1L

5.1.4 Equipment

Manufacturer: unknown
Model: unknown
Model number: unknown

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 1 h.

Detection is described in the following reference protocol:
Citation: Proteomics,5, page(s) 4264-4273 (2005).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

GS-710 calibrated densitometer scanner

6.1.2 Name of equipment

Manufacturer: Bio-rad
Model: GS-710 calibrated densitometer
Model number: unknown

6.1.3 Software

Manufacturer: GE Healthcare
Model: imagmaster
Model number: 5.0

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Default (vendor) parameters.

Configuration file: oocyte map

6.2 Acquisition Protocol

6.2.1 Image acquisition process

The stained gels were scanned at an optical resolution of 63.5 µm/pixel

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

oocyte map (format: TIFF)

7.1.2 Dimensions

Width: 85 px

Height: 85 px

7.1.3 Resolution

300 dpi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

Pig oocyte (1).tif

7.1.6 Standard image orientation

Yes

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