Western Blot Protoco-好.doc

Western Blot Protocol (8/19/2002) 1.Preparing the sample: -Cutting 20 um frozen sections to a slide, 3-5 of them; depends on the tissue size. (The slides can store at –80C for later study) -Air dry for 5 min or so. -Dissolution: Triple-detergent lysis buffer (from Sambrook Fritsch Maniatis): 50 mM Tris-HCL 150 mM NACL 0.1% SDS 100ug/ml PMSF 1ug/ml Aprorinin 1% NP-40 -100-200ul of lysis buffer is added to each slide, mix tissue with pipette tips -Scrape the mixture all from slide to a 1.5 ml tube. -Vortex well, put in to ice for 30-60 min -Spin 10 min at 14K rpm -Take the supernatant into a new tube 2. SDS(SDS-Polyacrylamide Gel Electrophoresis): -Choose the percentage of SDS-Polyacrylamide Gel according to protein molecular size. Acrylamide concentration (%) Linear range of separation (KD) 12-43 16-68 36-94 57-212 -Add 10-20ul of sample to same amount of 2X loading buffer (1:1), mix well, and include Protein Marker. 2X Loading buffer: 100 mM Tris-HCL (pH6.8) 200 mM DTT 4% SDS 0.2% Bromophenol blue 20% glycerol (DTT should be added just before the buffer is used, from 1M stock) -Boiling for 5-10 min -Preparing the GEL: mount the gel in the electrophoresis apparatus. -Add Tris-glycine electrophoresis running buffer to the top and bottom reservoirs. Tris-glycine Running Buffer: 25 mM Tris 250 mM glycine (electrophoresis grade) (pH8.3) 0.1% SDS -Remove any bubbles -Loading up 20-40ul of each sample in a predetermined order into the bottom of wells. -Attach the electrophoresis apparatus to an electric power supply (the positive elecrode should be connected to the bottom buffer reservoir) -Running at 200V for 45’-60’ -Remove the glass plates from the electrophoresis apparatus and place them on a paper towel. Using a spatula, pry the plates apart. -Mark the orientation of the gel by cutting a corner from the bottom of the gel. -Remove the gel from the electrophoresis apparatus and incubate it in Western Transfer Buffer for approximately 10 min to remove detergent. 3.