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Name: Reference map of multiple myeloma cells

Description: A two-dimensional gel electrophoresis (2DE) reference map of human multiple myeloma (MM) proteome is described here. Spots of 517 corresponding to 268 different proteins were identified on 2DE gels from plasma cells isolated from eight newly diagnosed MM patients. Proteins were classified into different categories based on their molecular functions and biological processes. This 2DE map of MM proteins will be an invaluable tool for further proteomics research that investigates proteomic changes associated with biomarker identification and carcinogenesis analysis of multiple myeloma.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2008-10-01

1.1.2 Responsible person or role

Affiliation: jinan university

(i) Name: jnnan university

(ii) Postal address: Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China
2Guangdong Provincial Key Laboratory of Bioengineering Pharmaceutics / National Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China

(iii) Email address: tgfeng@jnu.edu.cn

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: multiple myeloma reference map
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 8 M urea, 2% CHAPS, 20 mM DTT and 0.5% IPG buffer

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: IPGs NL pH 3-10, Linear pH4-7
Model number: 17-6001-15, 17-6001-13
Batch number: unknown

3.2.3 Physical dimensions

X: 130 mm
Y: 3 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

linear pH 4 - 7

3.2.5 Acrylamide concentration

4% %

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

Lane 1

  • Sample: multiple myeloma reference map
  • Volume of sample: 130 µg
  • Loading buffer: 8 M urea, 2% CHAPS, 20 mM DTT and 0.5% IPG buffer
  • Volume of loading buffer: 250 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

8 M urea, 2% CHAPS, 20 mM DTT and 0.5% IPG buffer

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 30 V, 10.00 h

Hold: 1000 V, 1 h

Hold: 8000 V, 8 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

Tris-HCL (50mM) pH 8.4

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: GE healthcare
Model: GE healthcare
Model number: Immobiline strip tray

4.1.5 Protocol

Temperature: 20 °C.

Duration: 4 h.

Protocol:
Equilibrate IPGstrips prior to SDS PAGE :
Equilibration Solution: 6M Urea 2% SDS, 50mM Tris.HCl pH 6.8, 30% Glycerol,
0.002% Bromophenol blue

Equilibration Solution can be aliquot and kept in the –20oC, DTT or Iodoacetamide (IAA)
must be added immediately before use.

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

Urea (6 M), glycerol (30% v/v), SDS (2% w/v) and DTT (2% w/v)

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: GE healthcare
Model: GE healthcare
Model number: Immobiline strip tray

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol Blue

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: GE healthcare
Model: GE healthcare
Model number: Immobiline strip tray

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,6, page(s) 2422 (2006).
URL: http://www3.interscience.wiley.com/cgi-bin/fulltext/112511043/PDFSTART

3.2.3 Physical dimensions

X: 140 mm
Y: 160 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 10 - 200 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: rehydration loading.

3.3 Protocol

3.3.1 Buffers

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

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 80 V, 4 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Silver staining

5.1.2 Direct detection agents

1.Fixation 30 Ethanol 100 ml 600 ml
or O/N Acetic Acid 25 ml 150 ml
2.Incubation 30 Ethanol 75 ml 450 ml
or O/N Na Acetate 10.25 g 61.5 g
anhydrous
Na Thiosulfate 0.5 g / *0.79 g 3 g / *4.71 g
3.Washing 3 x 10 dd H20
4.Silver Nitrate 40 Silver Nitrate 0.25 g 1.5 g
Formaldehyde 50 ul 300 ul
5.Development 15 Na-carbonate 6.25 g 37.5 g
Formaldehyde 25 ul 150 ul
6.Stop Solution 10 EDTA-Na.2H2O 3.65 g 21.9 g
7.Washing 3 x 5 ddH2O
8.Preservation 60 87% Glycerol 11.5 ml 69 ml
Ethanol 75 ml 450 ml

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

Detection is described in the following reference protocol:
Citation: Proteomics,6, page(s) 2422 (2006).
URL: http://www3.interscience.wiley.com/cgi-bin/fulltext/112511043/PDFSTART

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: Image Scanner (GE healthcare, Uppsala, Sweden)
Model number: 22771

6.1.3 Software

Manufacturer: GE healthcare
Model: Labscan
Model number: 2.0

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

Analytical gels were scanned on an Image Scanner (GE healthcare, Uppsala, Sweden) at 300 dpi with 12-bit gray scale levels in tagged image file format (TIFF), and images were analyzed using the ImageMaster 2D Platinum (GE healthcare, Uppsala, Sweden). All gels in the analyses were scanned with identical parameters. In order to perform differential analysis of the 2D gels with the ImageMaster 2D Platinum software, the individual spots of each gel were detected by their boundaries, and the spot volume corresponding to the protein abundance was calculated automatically. Each spot intensity volume was processed by background subtraction and total spot volume normalization; the resulting spot volume percentage was used for comparison. Manual editing of the gels was necessary especially in the higher molecular weight region. Only protein spots that were reproducibly different in all three experiments by at least a factor of two were considered to be significant and were excised from gels for analysis by MS.

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

MM (format: TIFF)

7.1.2 Dimensions

Width: 1049 px

Height: 1027 px

7.1.3 Resolution

300 dpi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

D:\重要文件\proteomics dataset draft\data to submit to World\Fig.1A.tif

7.1.6 Standard image orientation

Yes

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