Name: N/A Description: N/A Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2011-10-10 1.1.2 Responsible person or role Affiliation: FLETCHER LAB , SCHOOL OF MEDICINE ,LOMA LINDA UNIVERSITY (i) Name: WILSON ARUNI, FRANCIS ROY and HANSEL .M.FLETCHER (ii) Postal address: DIVISION OF MICROBIOLOGY AND MOLECULAR GENETICS SCHOOL OF MEDICINE LOMA LINDA UNIVERSITY LOMA LINDA -CA-92354,USA (iii) Email address: waruni@llu.edu 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Filifactor alocis -PROTEOME 2. Sample type: Test sample 3. URL: http://iai.asm.org/cgi/reprint/79/10/3872 Link: http://iai.asm.org/cgi/reprint/79/10/3872 2.1.2 Loading buffer 1. standard loading buffer for 2D gel electrophoresis 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 manufactured using the following reference protocol: Citation: Poznanovic, S., G. Schwall, H. Zengerling, and M. A. Cahill. 2005. Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 26:3185–3190.,26, page(s) 3185-3190 (2005). URL: http://iai.asm.org/cgi/reprint/79/10/3872 Link: http://iai.asm.org/cgi/reprint/79/10/3872 3.2.3 Physical dimensions X: 7 cm Y: 3 cm Z: 1 cm 3.2.4 Physiochemical property range and distribution linear pH 3 - 10 3.2.5 Acrylamide concentration 10 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 3.2:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Filifactor alocis -PROTEOME * Volume of sample: 5 µL * Loading buffer: standard loading buffer for 2D gel electrophoresis * Volume of loading buffer: 15 µL Loading method: well loading. 3.3 Protocol 3.3.1 Buffers 2D-REHYDRATING BUFFER 3.3.2 Electrophoresis conditions Running temperature: room temperature (no cooling device). Hold: 300 V, 3 h Hold: 3500 V, 5 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer EQUILIBIRATION BUFFER 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: BIORAD Model: BIORAD-PROTEAN ISOELECTRIC FOCUSING Model number: PROTEAN 4.1.5 Protocol 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: Poznanovic, S., G. Schwall, H. Zengerling, and M. A. Cahill. 2005. Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 26:3185–3190.,26, page(s) 3185-3190 (2005). URL: http://iai.asm.org/cgi/reprint/79/10/3872 Link: http://iai.asm.org/cgi/reprint/79/10/3872 3.2.3 Physical dimensions X: 7 cm Y: 5 cm Z: 1 cm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 10 - 250 kDa 3.2.5 Acrylamide concentration 10% % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 3.2:1 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 LOADING BUFFER-1 3.3.2 Electrophoresis conditions Running temperature: room temperature (no cooling device). Hold: 140 kV, 50 min 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents COOMASSIE SIMPLY BLUE STRAINING 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: BIORAD Model: X CELL SURE-LOCK Model number: BIORAD 5.1.5 Direct detection protocol Detection is described in the following reference protocol: Citation: Poznanovic, S., G. Schwall, H. Zengerling, and M. A. Cahill. 2005. Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 26:3185–3190.,26, page(s) 3185-3190 (2005). URL: http://iai.asm.org/cgi/reprint/79/10/3872 Link: http://iai.asm.org/cgi/reprint/79/10/3872 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment camera 6.1.2 Name of equipment Manufacturer: UVP Model: LAUNCH VISION WORK LS Model number: UVP 6.1.3 Software Manufacturer: UVP Model: LAUNCH VISION WORKS LS Model number: LAUNCH VISION WORKS LS 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 DEFAULT COMPANY SETTINGS FOR 2D PAGE IMAGING 6.2.2 Reference to gel matrix There is only one gel in this document.