将SDS-PAGE胶片中的蛋白质转印到膜上.docVIP

Western Blot Analysis – Part A 吳書瑋 2010//29 洪彩霞審核 2010/06/29 將SDS膠片中的蛋白質轉印到膜上”protein extraction, concentration determination and SDS” SOP. Materials and equipments: (1) Transfer tank (Hoefer TE 22) Specifications Max. wattage 50 W Max. voltage 100 V Max. amperage 500 mA Max. temperature 45 °C liters buffer required, depending on the number of cassettes in place 最多一次可轉印四片膠的蛋白質到四片膜上 Environmental temp: 4–40 °C PVDF membrane (Bio-Rad Cat. No. 162-0177) filter paper (Bio-Rad Cat. No. 1703967) Transfer buffer: 25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3, 2 liters 25 mM Tris (波仕特amresco Cat. No. 0826-1KG) (FW 121.1) 6.0 g 192 mM Glycine (仲郡 AppliChem Cat. No. A1123.2500) (FW 75.07) 28.8 g 0.1% (3.5 mM)SDS (波仕特amresco Cat. No. 0227-1KG) (FW 288.4) 2.0 g Dissolve in 1.5 liters distilled water. Add 400ml methanol (仲郡 Cat.No.ME0316). Bring to 2 liters with distilled water. (5) Fisher Stirring Hotplate Procedures: (1) Prepare 9 X 6 cm of PVDF membrane(s) and 10 X 7 cm filter paper (2 pieces of filter paper for each membrane). (2) Pre-wet PVDF membranes in methanol. Then, soak all membrane and filter paper in transfer buffer for 2–5 minutes. (3) Assemble the transfer stack so that proteins will migrate from gel toward the membrane. (4) Place one 3 mm-thick sponge on the opened submersed cassette and press gently until all air is expelled. Place one sheet of filter paper on the sponge, and then place the membrane on the blotting paper. 將膠片放在膜上面。Gently roll a 15 ml test tube over the gel to expel trapped air between the membrane and gel. Cover the gel with a sheet of filter paper, and then place a sponge of the proper thickness (see the diagram below), again pressing gently to expel trapped air. Assemble the cassette in a tray containing transfer buffer about 3 cm deep. (7) Close the cassette and press lightl