Gel: Reference map for Corynebacterium glutamicum ATCC14067, pH 4.5-5.5 (html display)

Name: Reference map for Corynebacterium glutamicum ATCC14067, pH 4.5-5.5

Description: Cytoplasmic protein extract from cells of Corynebacterium glutamicum ATCC14067, pH 4.5-5.5
Reference: PROTEOMICS (2007) 7(23): 4317-4322 (doi:10.1002/pmic.200700269)

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2003-04-01

1.1.2 Responsible person or role

Affiliation: Hokkaido University

(i) Name: Prof. Dr. Atsushi Yokota

(ii) Postal address: Laboratory of Microbial Physiology
Research Faculty of Agriculture
Hokkaido University
Sapporo, Hokkaido 060-8589, Japan

(iii) Email address: yokota@chem.agr.hokudai.ac.jp

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

1. Sample name: Cytoplasmic protein extact from cells of Corynebacterium glutamicum ATCC14067
2. Sample type: Test sample
3. URL: http://dx.doi.org/10.1002/pmic.200700269

2.1.2 Loading buffer

8 M urea, 4% w/v CHAPS, 65 mM DTT, 40 mM Tris-HCl (pH 8.0), 2% v/v IPG buffer, and a trace of 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 Corp.
Model: Immobiline DryStrip 4.5-5.5
Model number: 17-6001-85
Batch number: unknown

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 4.5 - 5.5

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 substance

3.2.8 Gel lane

3.2.9 Sample application

Lane 1

Sample: Cytoplasmic protein extact from cells of Corynebacterium glutamicum ATCC14067
Volume of sample: 100 µg
Loading buffer: 8 M urea, 4% w/v CHAPS, 65 mM DTT, 40 mM Tris-HCl (pH 8.0), 2% v/v IPG buffer, and a trace of bromophenol blue
Volume of loading buffer: 400 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 60 V, 0.1 h

Hold: 150 V, 1 h

Hold: 500 V, 2 h

Gradient: 500-3500 V, 18 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

Buffer A: 6 M urea, 30% v/v glycerol, 1% w/v SDS, 16.25 mM DTT, 50 mM Tris-HCl, pH 6.8.
Buffer B: buffer A in which DTT was replaced with 45 mg/mL iodoacetamide.

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: Multiphor II
Model number: 18-1018-06

4.1.5 Protocol

Protocol:
10 min in Buffer A and then 10 min in Buffer B

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 purchased precast.

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: ExcelGel XL SDS 12-14
Model number: 17-1236-01
Batch number: unknown

3.2.3 Physical dimensions

X: 245 mm
Y: 180 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 12 - 116 kDa

3.2.5 Acrylamide concentration

12-14 % (unknown)

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

3.2.9 Sample application

Loading method: IPG_transfer.

3.3 Protocol

3.3.1 Buffers

0.12 M Tris Acetate (pH 6.6), 1g/L SDS

3.3.2 Electrophoresis conditions

Running temperature: room temperature (no cooling device).

Hold: 1000 V, 45 min

Hold: 1000 V, 4 min

Hold: 1000 V, 160 min

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

SYPRO Ruby Protein Gel Stain (Bio-Rad Laboratories, Inc.)

5.1.3 Additional reagents and buffers

fixing solution: 10% (v/v) Ethanol, 7% (v/v) Acetic acid

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Protocol:
according to the instruction from the manufacturer:
http://wolfson.huji.ac.il/purification/PDF/Stains/BioRadSyproRubinProtStains.pdf

Detection is described in the following reference protocol:
Citation: not provided.
URL: http://www.proteomic.org/html/staining.html

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

fluorescent scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: FluorImager 595
Model number: 63001545

6.1.3 Software

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: ImageQuant
Model number: unknown

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

unknown

6.2 Acquisition Protocol

6.2.1 Image acquisition process

unknown

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

CORGL-ATCC14067-4.5-5.5 (format: JPEG)

7.1.2 Dimensions

Width: 2008 px

Height: 1606 px

7.1.3 Resolution

300 dpi

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

http://world-2dpage.expasy.org/0001/html/data/gifs/yokota_0001/CORGL-ATCC14067-4.5-5.5.jpg

7.1.6 Standard image orientation

Yes