Name: Mytilus edulis sperm

Description: 2-DE proteome map and identification of highly expressed protein spots in sperm of Mytilus edulis

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2007-12-01

1.1.2 Responsible person or role

Affiliation: University of Vigo

(i) Name: Angel P. Diz

(ii) Postal address: Department of Biochemistry, Genetics and Immunology
Faculty of Biology
University of Vigo
Campus Universitario de Vigo s/n
36310 Vigo
Spain
Tel. +34 986813828
Population Genetics and Cytogenetics Group (XB2)
http://webs.uvigo.es/genxb2

(iii) Email address: angel.p.diz@uvigo.es

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Sperm from Mytilus edulis
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1% IPG

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 3-10 NL, 24 cm
Model number: 17-6002-45
Batch number: unknown

3.2.3 Physical dimensions

X: 240 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

4 %

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: Sperm from Mytilus edulis
  • Volume of sample: 300 µg
  • Loading buffer: 7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1% IPG
  • Volume of loading buffer: 450 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1% IPG

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 500 V, 2 h

Gradient: 500-1000 V, 3 h

Hold: 8000 V, 11 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

50 mM Tris-HCl (pH 8.8), containing 2% (w/v)
SDS, 1% (w/v) dithiothreitol (DTT), 6 M urea and 30% (w/v) glycerol

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

[50 mM Tris-HCl (pH 8.8), containing 2% (w/v)
SDS, 4% (w/v) iodoacetamide, 6 M urea and 30% (w/v) glycerol

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

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 receipe:

Acrylamide/bis (30% T, 37.5:1 líquid)
H20 mQ
1.5 M Tris-HCL, pH 8.8
10% SDS
10% ammonium persulfate (fresh)
TEMED

3.2.3 Physical dimensions

X: 22 cm
Y: 27 cm
Z: 0.1 cm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 200 - 10 kDa

3.2.5 Acrylamide concentration

12.5 %

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 load on the top of 2-DE gel.

3.3 Protocol

3.3.1 Buffers

1× SDS electrophoresis buffer

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 100 V, 45 min

Hold: 500 V, 6 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Silver staining

5.1.2 Direct detection agents

N/A

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: 20 °C.

Duration: 5 h.

Detection is described in the following reference protocol:
Citation: Anal. Chem.,68, page(s) 103-112 (1985).
URL: http://pubs.acs.org/doi/abs/10.1021/ac950914h

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

laser scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare
Model: ImageScanner (GE Healthcare)
Model number: Unknown

6.1.3 Software

Manufacturer: GE Healthcare
Model: ImageMaster Platinum
Model number: 5

6.1.4 Calibration

No (manual)

6.1.5 Equipment specific parameters

Default (vendor) parameters.

6.2 Acquisition Protocol

6.2.1 Image acquisition process

We have followed manufacturer instructions

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

Mytilus edulis sperm (format: TIFF)

7.1.2 Dimensions

Width: 1000 px

Height: 1000 px

7.1.3 Resolution

300 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

Mytilus sperm gel World 2DPAGE large format.tif

7.1.6 Standard image orientation

Yes

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