第四章 SDS-PAGE

1、Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinforma

2、tics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 凝胶浓度凝胶浓度T（C=2.6） 分子质

3、量范围分子质量范围（kDa） 凝胶浓度凝胶浓度T（C=5） 分子质量范围分子质量范围（kDa） 5 5101015152020 25-20010-70 50405 5101015152020 60-17020-10010- 50 5 - 40Bioinformatics, 2010-2011, Shanxi Agricultural University 或高于或高于2525都不能被使用。都不能被使用。Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agric

4、ultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 标准蛋白分子量标准蛋白分子量未未知知蛋蛋白白在一定的凝胶浓度下，在一定的凝胶浓度下，多肽链分子量的对数与多肽链分子量的对数与多肽链的相对迁移率成多肽链的相对迁移率成线性关系，所以可以通线性关系，所以可以通过标准曲线求未知多肽过标准曲线求未知多肽链分子量链分子量。 相对迁移率相对迁移率Bioinformatics, 2010-2011, Shanxi Agricultural University 分析计算分析计算= 蛋白样品距加样端迁移距离c

5、m溴酚蓝区带中心距加样端距离cm 相对迁移率每个蛋白标准的分子量对数对它的相对迁移率作图得标准曲线，量出未知蛋白的迁移率即可测出其分于量，这样的标难曲线只对同一块凝胶上的样品的分子量测定才具有可靠性。Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011,

6、 Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 贮液贮液 凝胶浓度凝胶浓度(c=2.6(c=2.6) )单体贮液（单体贮液（mLmL）缓冲液贮液（缓冲液贮液（mLmL）双蒸水（双蒸水（mLmL）1010过硫酸铵（过硫酸铵（uLuL）TEMED TEMED （uLuL）5.3 10.04.5100100T=5.1 6.710.03.11001009.010.0 0.8100100T=10.2 T=7.7 Bioinformatics, 2010-2011, Shan

7、xi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 贮液贮液 凝胶终浓度凝胶终浓度单体贮液（单体贮液（mLmL）浓缩胶缓冲液贮液（浓缩胶缓冲液贮液（mLmL）分离胶缓冲液贮液（分离胶缓冲液贮液（mLmL）双蒸水（双蒸水（mLmL）1010过硫酸铵（过

8、硫酸铵（uLuL）TEMED TEMED （uLuL）3.4 2.4-0.2141010 T=3 T=5 5-7.50.317.21010T=10 10-7.50.312.21010T=15 T=20 15- 7.50.37.21010 20-7.50.32.21010 Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 贮液贮液 凝胶终浓度凝胶终浓度单体贮液（单体贮液（mLmL）浓缩胶缓冲液贮液（浓缩胶缓冲液贮液（

9、mLmL）分离胶缓冲液贮液（分离胶缓冲液贮液（mLmL）甘油（甘油（mlml）双蒸水（双蒸水（mLmL）1010过硫酸铵（过硫酸铵（uLuL）TEMED TEMED （uLuL）3.4 2.4-1.00.2141010 T=3 T=5 5-7.50.30.317.21010T=10 10-7.50.30.312.21010T=15 T=20 15- 7.50.30.37.21010 20-7.50.30.32.21010 Bioinformatics, 2010-2011, Shanxi Agricultural University 水平式电泳装置水平式电泳装置Bioinformatics,

10、 2010-2011, Shanxi Agricultural University -3-3二硫二硫 苏糖醇或苏糖醇或4 4-5-5Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 低分子量标准蛋白试剂盒低分子量标准蛋白试剂盒: :兔磷酸化酶兔磷酸化酶B MW=97,400 B MW=97,400

11、牛血清白蛋白牛血清白蛋白 MW=66,200 MW=66,200 牛碳酸酐酶牛碳酸酐酶 MW=31,000MW=31,000 胰蛋白酶抑制剂胰蛋白酶抑制剂 MW=20,100MW=20,100 鸡蛋清溶菌酶鸡蛋清溶菌酶 MW=14,400MW=14,400Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 将电泳仪的正极与下槽连接，负极与上槽连接，打将电泳仪的正极与下槽连接，负极与上槽连接，打开电泳仪开关，开始时电流

12、为开电泳仪开关，开始时电流为10 mA10 mA，待样品进入分，待样品进入分离胶后，将电流调至离胶后，将电流调至20-30 mA20-30 mA，当溴酚蓝染料距硅，当溴酚蓝染料距硅胶框胶框1cm1cm时，停止电泳，关闭电源。时，停止电泳，关闭电源。Bioinformatics, 2010-2011, Shanxi Agricultural University Staking gelSeparating gel垂直电泳装置垂直电泳装置Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-20

13、11, Shanxi Agricultural University 水平式电泳装置水平式电泳装置Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 201

14、0-2011, Shanxi Agricultural University 固定固定试剂试剂 时间时间 500ml500ml乙醇乙醇100ml100ml冰醋酸冰醋酸400ml400ml蒸馏水蒸馏水. .浸泡浸泡75ml75ml乙醇乙醇17g17g醋酸钠醋酸钠1.25ml251.25ml25戊二醛戊二醛0.5g0.5g硫代硫酸钠硫代硫酸钠.5H.5H2 2O O用用ddddH2O溶解后加至溶解后加至250ml30mins30mins步骤步骤至少至少30mins30mins漂洗漂洗用蒸馏水漂洗用蒸馏水漂洗3 3次次5mins/5mins/次次Bioinformatics, 2010-2011,

15、Shanxi Agricultural University 银染银染试剂试剂 时间时间 0.25g0.25g硝酸银硝酸银50ul50ul甲醛甲醛用用ddHddH2 2O O溶解后溶解后加至加至250ml250ml显色显色6.25g6.25g碳酸钠碳酸钠25ul25ul甲醛甲醛用用ddH2O加至加至250ml2-10mins2-10mins视蛋白视蛋白带显示深棕色带显示深棕色步骤步骤20mins20mins终止终止3.65gEDTA-Na3.65gEDTA-Na2 2.2.2H2O用蒸馏水加至用蒸馏水加至250ml250ml10mins10mins漂洗漂洗保存保存用蒸馏水漂洗用蒸馏水漂洗3 3

16、次次5mins/次次250250甘油用水加至甘油用水加至250ml250ml30mins取出晾干取出晾干Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 涂抹痕迹可能由于多种原因引起，但

17、是最常见的原因是聚丙烯酰胺凝胶倒胶的不均匀或者上样量过大。这块胶倒胶不正确，在上部胶倒入制胶槽之前就已经发生聚合。首先倒入的胶聚合发生的太快了。与其重新倒胶，不如停止倒胶重新准备新的聚丙烯酰胺混合液，然后将新的胶倒入旧胶的上方。但是显然这种连接不是非常牢固。（我推荐重新倒胶，制胶很关键，如果你后续还要做blot，胶没有跑好浪费太多了！） Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics

18、, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 条纹拖尾条纹拖尾例例1 1Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformat

19、ics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-

20、2011, Shanxi Agricultural University 部分前端指示剂部分前端指示剂- -例例1 1Bioinformatics, 2010-2011, Shanxi Agricultural University 部分前端指示剂部分前端指示剂- -例例2 2Bioinformatics, 2010-2011, Shanxi Agricultural University 部分前端指示剂部分前端指示剂- -例例3 3Bioinformatics, 2010-2011, Shanxi Agricultural University 症状症状5-5-条带仅在凝胶顶部条带仅在凝胶顶

21、部过早终止电泳过早终止电泳例例1 1 Bioinformatics, 2010-2011, Shanxi Agricultural University 过早终止电泳过早终止电泳- -例例2 2Bioinformatics, 2010-2011, Shanxi Agricultural University 症状症状6-6-混杂因素混杂因素扭曲的条带扭曲的条带Bioinformatics, 2010-2011, Shanxi Agricultural University 加样孔不成形加样孔不成形 Bioinformatics, 2010-2011, Shanxi Agricultural U

22、niversity Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 多种症状多种症状- -例例1 1 Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural