Name: Proteomic analysis of rat cerebellum Description: To study the biochemistry of the cerebellar cortex by using proteomic methods. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2008-11-15 1.1.2 Responsible person or role Affiliation: Centre for Cellular and Molecular Biology (i) Name: Purnima Bhargava (ii) Postal address: Uppal Road, Tarnaka, Hyderabad-500 007 India (iii) Email address: purnima@ccmb.res.in 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Cerebellum 2. Sample type: Test sample 2.1.2 Loading buffer 1. 7M UREA, Thiourea 2M, CHAPS 4%, 18mM Tris (pH 8.0), Trizma 14mM, Triton X-100 0.2%, DTT 50mM, IPGBuffer 2% and trace amount 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 AB Model: Immobiline Dry Strip pH 3-10 NL Model number: 17-6001-15 Batch number: 10013248 3.2.3 Physical dimensions X: 128 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution sigmoidal pH 3 - 10 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 substances 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Cerebellum * Volume of sample: 1 mg * Loading buffer: 7M UREA, Thiourea 2M, CHAPS 4%, 18mM Tris (pH 8.0), Trizma 14mM, Triton X-100 0.2%, DTT 50mM, IPGBuffer 2% and trace amount bromophenol blue. * Volume of loading buffer: 300 µL * Volume of mixture loaded in the lane: 250 µL Loading method: cup loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 100 V, 1.30 h Hold: 300 V, 2 h Hold: 500 V, 2 h Hold: 1000 V, 2 h Hold: 8000 V, 15.00 h Hold: 8000 V, 10.00 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer Buffer I- 6M urea, 0.375M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol, 2% DTT Buffer II- Buffer I with 2.5% iodoacetamide instead of DTT 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 25 min. Protocol: 25 minutes in buffer I 25 minutes in buffer II 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 Composed of 2 sub-gels (Stacking gel, Running gel). 3.2.2 Gel manufacture Gel was manufactured using the following reference protocol: Citation: not provided. URL: http://www.ncbi.nlm.nih.gov/pubmed/5432063 Link: http://www.ncbi.nlm.nih.gov/pubmed/5432063 3.2.3 Physical dimensions Stacking gel: X: 140 mm Y: 10 mm Z: 1.5 mm Running gel: X: 140 mm Y: 140 mm Z: 1.5 mm 3.2.4 Physiochemical property range and distribution Stacking gel: No distribution in a Stacking gel. Running gel: logarithmic apparent molecular mass 250 - 18 kDa 3.2.5 Acrylamide concentration Stacking gel: 5% % Running gel: 12% % 3.2.6 Acrylamide : Crosslinker ratio Stacking gel: Crosslinker: Bisacrylamide Ratio: 29:1 Running gel: Crosslinker: Bisacrylamide Ratio: 29:1 3.2.7 Additional substances in gel Stacking gel: No additional substances Running gel: No additional substances 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: IPG_transfer. 3.3 Protocol 3.3.1 Buffers Tris-glycin-SDS (25mM-198mM-0.1%w/v)pH 8.3 3.3.2 Electrophoresis conditions Running temperature: 25 °C Hold: 10 mA, 12 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents Coomassie briliant blue R250 5.1.3 Additional reagents and buffers Solution1: CBB 0.5g, isopropanol 250ml, Glacial acetic acid 100ml, Water 650 ml Solution2: CBB 0.05g, isopropanol 100ml, Glacial acetic acid 100ml, Water 800 ml Solution3: CBB 0.02g, Glacial acetic acid 100ml, Water 900 ml Solution4: Glacial acetic acid 100ml, Water 900 ml 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Protocol: 1.At the end of second dimension gels were washed in MQ for 15 minutes. 2.Soaked in Solution1 and heated for 1.20min and cooled for 30minutes 3.Washed in MQ for 5 minutes. 4.Soaked in Solution2 and heated for 1.20min and cooled for 30minutes 5.Washed in MQ for 5 minutes. 6.Soaked in Solution3 and heated for 1.20min and cooled for 30minutes 7.Washed in MQ for 5 minutes. 8.Soaked in solution 4 and heated for 1.20min and cooled for 30minutes 9.Above is repeated till the proper distaining. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment camera 6.1.2 Name of equipment Manufacturer: Syngene Model: Dyversity Model number: Unknown 6.1.3 Software Manufacturer: Syngene Model: GeneSnap Model number: 6.08 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 According to manufacturer's protocol 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) cereb21rat (format: TIFF) 7.1.2 Dimensions Width: 1141 px Height: 1276 px 7.1.3 Resolution 72 ppi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location cereb21rat.tif Link: /download/21/Usf1f3Qa/ 7.1.6 Standard image orientation Yes