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1、非变性非还原蛋白凝胶电泳A: Loading buffer for gel:From Dr. Chen Q. (6X)Glycerol20%Bromophenol bluesmall bit (0.1%)0.2%Do not boil the samples!B: SDS-PAGE Separating Gel12%DH2O10.2ml3.41.5M Tris (pH8.8)7.50ml2.50Acrylamide (29:1, 30% )12.0ml4.0AP(10%)300ul100TEMED12ul4Total Volume30ml10mlC: SDS-PAGE Stacker GelT
2、ris-HCl 0.5M (pH6.8)0.625mldH2O1.55 mlAcrylamide (29:1, 30%)0.325 mlAP 10%12.5ulTEMED2.5 ulTotal Volume2.5 mlD: SDS Running buffer for 1 liter (10X) pH 8.3Tris30 gGlycine188 gSDS10 gBring up to 1 L with dH2ODilute to 1x with dH O when used.2E: PAGE Program(1) Assemble the gel kit(2) Pour the separat
3、ing gel mixture between the plates ( about 2/3 of the way up) and overlay with water or isobutanol (better than water,异丁醇)(异丙醇也可)(3) Leave to set and then wash off the water(4) Pour on the stacking gel mix and insert the comb then leave to set(5) Transfer to the running apparatus and pour in the run
4、ning buffer(6) Remove the comb and load the sample (20 胆/lane)(7) Run the gel 50V through the stacking gel, then 100V through the separating gel.D: staining:凝胶用考马斯亮蓝染液室温温和摇动染色0.5个小时，然后经脱色液脱色2小时，去离子水 洗净余液即可。考马斯亮蓝染色液（见16 5）50% (v/v)0* 05% (v/v)10% (v/v)40%甲醇苦马斯亮蓝 R-250 (Bio-Rad 或 Pierce)乙酸H2O用去离子蒸憎水配制脱色液（见10*5）7% v/v)乙酸5% v/v)甲醇88%HQ
