Western Blot Protoco-好.docVIP

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