Two Dimensional Gel Electrophoretic Analysis for the Human Plasma

Overview    This protocol is a detail description of the laboratory procedure in performing 2D gel electrophoresis for illustrating the protein profile of the human plasma proteome.      Material  1.Peripheral whole blood. 2.Centrifuge machine 3.Spectrophotometer 4.Sample solubilization solution: (1).25.5 g urea + 50 ml distilled H2O (2).0.385 g DTT (3).2 ml NP-40 (4).250 £gl carrier ampholyte (pH 3 ¡V 10) (5).Trace amount Bromophenol blue 5.IPG strip 6.Mineral oil 7.Rehydration & IEF unit(BioRad) 8.Equilibration buffer: (1).Tris (pH: 8.8; 50 mM): 0.335 ml (2).Urea: 3.604 g (3).Glycerol: 3.15 ml (4).SDS: 0.2 g (5).Bromophenol blue (trace) (6).Make up to 10 ml with distilled H2O 9.100 mg DTT (Dithiothreitol) 10.250 mg IAA (Iodoacetamide) 11.Running buffer: (1).Tris (ultrapure) 30.285 g (2).SDS 144.134 g (3).Glycine (ultrapure) 10 g (4).Make up to 1000 ml with distilled H2O 12.Polyacrylamide gel: (1).Distilled H2O: 24.7 ml (2).30% acrylamide: 22.1 ml (3).1.5 M Tris (pH: 8.8): 16.9 ml (4).10% SDS: 0.65 ml (5).10% ammonium persulfate: 0.65 ml (6).TEMED: 0.026 ml 13.0.5% agarose gel 14.Protein marker (BioRad) 15.The vertical SDS-PAGE unit(BioRad) 16.The glass staining basin 17.The fixation solution: (1).100 ml Ethanol (2).25 ml Acetic acid glacial (3).Make up to 250 ml with distilled H2O 18.The sensitizing solution: (1).75 ml Ethanol (2).1.25 ml Glutardialdehyde (25% w/v) (3).10 ml Sodium thiosulphate (5% w/v) (4).17 g Sodium acetate (5).Make up to 250 ml with distilled H2O (6).Distilled water 19.The developing solution: (1).6.25 g Sodium carbonate (2).0.05 ml Formaldehyde (37% w/v) (3).Make up to 250 ml with distilled H2O 20.The stopping solution: (1).3.65 g EDTA-Na2 ¡D2 H2O (2).Make up to 250 with distilled H2O         Procedure    1.The peripheral whole blood sample is collected in heparin tube. 2.Record the patients¡¦ name, chart no., and tube no. 3.Centrifuge at 4¢J 3000 rpm for 10 minutes. 4.Estimation of protein concentration by spectrophotometry i.e. checking OD595 (1).Mix the followings: 200£gl of BSA (Protein Assay Solution) 20£gl sample of human plasma 798 £gl H2O (2).Blank solution: 200£gl of BSA (Protein Assay Solution) 800£gl H2O From the ¡§O.D. vs Concentration¡¨ graph we can estimate the concentration of protein in the human plasma (x mg / 1 ml) 5.Volume of protein sample recruited: The amount of protein sample required for silver stain is 300£gg By this calculation: x mg / 1 ml = 300£gg / y £gl y £gl of the sample of human plasma is to be recruited in terms of its volume 6.Weigh 38 mg DTT and place it in the eppendorf tube. (Mix the sample and DTT first, and then add the buffer solution.) 7.Add buffer solutions to the sample. Ingredients of the sample solubilization solution: (1).25.5 g urea + 50 ml distilled H2O (2).0.385 g DTT (3).2 ml NP-40 (4).250 £gl carrier ampholyte (pH 3 ¡V 10) (5).Trace amount Bromophenol blue The amount of sample buffer required = 300 £gl - y £gl (protein sample) = z £gl 8.Load this mixture to the strip tray. 9.Place the IPG (immobilized pH gradient) strip into the reswelling tray with gel-surface facing downward. 10.Add wicks soaked with buffer solution on both electrodes to minimize the interfering effects of salts in the solution. 11.Cover the strip with 1 ml mineral oil to prevent evaporation & urea crystallization 12.Cover the reswelling tray with its lid and set the program for rehydration and IEF. Step 1: 250 volts, 15 min Step 2: 10000 volts, 3 hours Step 3: 60000 volts, 6 hours Step 4: 500 volts, hold *Duration: 12 hours 13.Harvest the IPG strip. 14.Rinse the IPG strip to remove the mineral oil. 15.Equilibration *1st Equilibration: (Soak the IPG strip in 5 ml of this equilibration solution for 30 minutes with shaking) 10 ml SDS equilibration buffer + 100 mg DTT (Dithiothreitol) *2nd Equilibration: (Soak the IPG strip in 5 ml of this equilibration solution for 30 minutes with shaking) 10 ml SDS equilibration buffer + 250 mg IAA (Iodoacetamide) *Equilibration buffer: (1).Tris (pH: 8.8; 50 mM): 0.335 ml (2).Urea: 3.604 g (3).Glycerol: 3.15 ml (4).SDS: 0.2 g (5).Bromophenol blue (trace amount) (6).Make up to 10 ml with distilled H2O 16.Meanwhile prepare the polyacrylamide gel: (1).Distilled H2O: 24.7 ml (2).30% acrylamide: 22.1 ml (3).1.5 M Tris (pH: 8.8): 16.9 ml (4).10% SDS: 0.65 ml (5).10% ammonium persulfate: 0.65 ml (6).TEMED: 0.026 ml 17.Add running buffer on the top edge of the polyacrylamide gel 18.After equilibration, the IPG strip is placed horizontally on top of the polyacrylamide gel 19.Cover the top edge with 0.5% agarose gel, and create a marker well on top of the gel 20.Load protein marker into the marker well. 21.Place the polyacrylamide gel set into the vertical SDS-PAGE tank. 22.Fill it with sufficient running buffer. 23.Cover the lid of the vertical SDS-PAGE tank. 24.Switch on the power supply. *Running conditions of 2D PAGE: (1).Current: 40 mA/gel (2).Voltage: 100 volts (3).Temperature: 8 -12¢XC (keep the system cool inside the refrigerator) (4).Duration: 6 hours 25.Harvest the polyacrylamide gel. 26.Place it into the staining basin. 27.The silver staining procedure: (1).Fixation (2).Sensitizing (3).Washing (4).Silver reaction (5).Washing (6).Developing (7).Stopping 28.Fixation (1).100 ml Ethanol (2).25 ml Acetic acid glacial (3).Make up to 250 ml with distilled H2O (4).Action time: 30 minutes 29.Sensitizing (1).75 ml Ethanol (2).1.25 ml Glutardialdehyde (25% w/v) (3).10 ml Sodium thiosulphate (5% w/v) (4).17 g Sodium acetate (5).Make up to 250 ml with distilled H2O (6).Action time: 30 minutes 30.Washing (1).Distilled H2O (2).3 times (3).Each for 5 minutes 31.Silver reaction (1).25 ml Silver nitrate solution (2.5% w/v) (2).Formaldehyde (37% w/v) (3).Action time: 20 minutes 32.Washing (1).Distilled H2O (2).2 times (3).Each for 1 minute 33.Developing (1).6.25 g Sodium carbonate (2).0.05 ml Formaldehyde (37% w/v) (3).Make up to 250 ml with distilled H2O (4).Stir vigorously to dissolve sodium carbonate (5).Action time: 2 ¡V 5 minutes 34.Stopping (1).3.65 g EDTA-Na2¡D2 H2O (2).Make up to 250 with distilled H2O (3).Action time: 10 minutes 35.Scan and file the results. 36.Identify the proteins by means of specific software.         Troubleshooting    1.Make sure the rehydration tray is placed horizontally. 2.Bubble-formation should be avoided when loading the strip into the tray. 3.The IPG strip should be stored under -20¢J, and should not be exposed to room temperature longer that 10 minutes. 4.Make sure there is no leakage of water from the vertical glass plates before loading polyacrylamide gel solution.         Reference    N. Leigh Anderson, Norman G. Anderson. The Human Plasma Proteome. History, character, and diagnostic prospects. Molecular & Cellular Proteomics 2002;1.11:845-67.