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. 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 Link: 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 Link: 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 Link: /download/25/wHjFDUkC/ 7.1.6 Standard image orientation Yes