Western Blot(免疫印迹法)实验方法步骤.docVIP

Western Blot(免疫印迹法)实验方法步骤 Western Blot(免疫印迹法) 主要包括以下4个基本步骤： n?????样品制备 n??? ?电泳分离 n??? ?蛋白的膜转移 n??? ?免疫杂交与显色――蛋白检测 溶液和试剂 n??? 1X 磷酸盐缓冲液(PBS) n??? Modified RIPA buffer Tris-HCl: 50 mM, pH 7.4 ; NP-40: 1% ;Na-deoxycholate: 0.25% ;NaCl: 150 mM ;EDTA: 1 mM ;PMSF: 1 mM ;Aprotinin, leupeptin, pepstatin: 1 microgram/ml each ;Na3VO4: 1 mM ;NaF: 1 mM n??? 1X SDS 样品缓冲液 62.5 mM Tris-HCl (pH 6.8 于 25°C), 2% w/v SDS, 10%甘油,50 mM DTT, 0.01% w/v溴酚蓝 n??? 转移缓冲液 25 mM Tris base, 0.2 M 甘氨酸, 20%甲醇 (pH 8.3) n??? 10X Tris缓冲盐 (TBS) 准备1L 10X TBS: 24.2 g Tris base, 80 g NaCl;用1N HCl调pH为 7.6 n??? 脱脂奶粉或BSA n??? 甲醇 n???