EMSA实验步骤.doc

EMSA实验步骤 Ⅰ.核蛋白的提取 1. Collect 6g leaf and grind with liquid nitrogen. 2. Transfer the powder into a homogenizing buffer and stir for over 20 min. 3. The mixture was filtrated with a Miracloth and collected the supernatant 4. The mixture was centrifuged at 3000 g for 20 min at 4°C. 5. Wash the pellet 3 times with low salt buffer, after washing centrifuge. 6. The pellet was suspend in a high salt buffer and centrifuge for 30 min at 12000 rpm. 7. Collect the supernatant and dialysis in low salt buffer for over 1 h. Ⅱ.标探针 DNA 4ul（50ng） KF 1ul （10U） dNTP（不含dCTP） 2ul 10×buffer 2ul dCTP* 2ul ddH2O 9ul tatol 20ul Ⅲ.制胶 6％的PAGE胶（一般制蛋白质电泳用的胶中的分离胶），注意不要加SDS。 Ⅳ.DNA binding 10×DNA binding buffer 80％甘油 63.5ul 5M NaCl 10ul 1M Tris-HCl pH7.5 10ul 0.1M MgCl2 10ul 0.25M EDTA 4ul 1M DTT 1ul ddH2O 1.5ul tatol 100ul DNA binding 体系： 蛋白质 5ug 10×binding buf 2ul add ddH2O to 16ul 室温放置15－20min 电压40V就,电泳两个小时左右，溴酚蓝刚好跑到胶的最底端，就可以停止电泳。 Ⅵ. 压膜和显影 用预先剪好的滤纸夹住胶，然后外面再包一层保鲜膜，压片，5个小时左右就可以冲洗了，也可以过夜。 Reagent homogenizing buffer 10 mM HEPES at pH 7.8, 10 mM KCl, 10 mM MgCl2, 5 mM EDTA, 1 mM DTT, 200 μM PMSF, 250 mM sucrose, 0.5% Triton X-100 low salt buffer 20 mM HEPES at pH 7.8, 20 mM KCl, 1.5 mM MgCl2, 25% glycerol, 200 (M EDTA, 500 (M DTT, 200 (M PMSF high salt buffer 20 mM HEPES at pH 7.8, 1.5 M KCl, 1.5 mM MgCl2, 25% glycerol, 200 (M EDTA, 500 (M DTT, 200 (M PMSF 1×binding buffer 10 mM Tris at pH 7.5, 5