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 凝膠濃度凝膠濃度T（C=2.6） 分子質(zhì)

3、量范圍分子質(zhì)量范圍（kDa） 凝膠濃度凝膠濃度T（C=5） 分子質(zhì)量范圍分子質(zhì)量范圍（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 標(biāo)準(zhǔn)蛋白分子量標(biāo)準(zhǔn)蛋白分子量未未知知蛋蛋白白在一定的凝膠濃度下，在一定的凝膠濃度下，多肽鏈分子量的對(duì)數(shù)與多肽鏈分子量的對(duì)數(shù)與多肽鏈的相對(duì)遷移率成多肽鏈的相對(duì)遷移率成線(xiàn)性關(guān)系，所以可以通線(xiàn)性關(guān)系，所以可以通過(guò)標(biāo)準(zhǔn)曲線(xiàn)求未知多肽過(guò)標(biāo)準(zhǔn)曲線(xiàn)求未知多肽鏈分子量鏈分子量。 相對(duì)遷移率相對(duì)遷移率Bioinformatics, 2010-2011, Shanxi Agricultural University 分析計(jì)算分析計(jì)算= 蛋白樣品距加樣端遷移距離c

5、m溴酚藍(lán)區(qū)帶中心距加樣端距離cm 相對(duì)遷移率每個(gè)蛋白標(biāo)準(zhǔn)的分子量對(duì)數(shù)對(duì)它的相對(duì)遷移率作圖得標(biāo)準(zhǔn)曲線(xiàn)，量出未知蛋白的遷移率即可測(cè)出其分于量，這樣的標(biāo)難曲線(xiàn)只對(duì)同一塊凝膠上的樣品的分子量測(cè)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 貯液貯液 凝膠濃度凝膠濃度(c=2.6(c=2.6) )單體貯液（單體貯液（mLmL）緩沖液貯液（緩沖液貯液（mLmL）雙蒸水（雙蒸水（mLmL）1010過(guò)硫酸銨（過(guò)硫酸銨（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 貯液貯液 凝膠終濃度凝膠終濃度單體貯液（單體貯液（mLmL）濃縮膠緩沖液貯液（濃縮膠緩沖液貯液（mLmL）分離膠緩沖液貯液（分離膠緩沖液貯液（mLmL）雙蒸水（雙蒸水（mLmL）1010過(guò)硫酸銨（過(guò)

8、硫酸銨（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 貯液貯液 凝膠終濃度凝膠終濃度單體貯液（單體貯液（mLmL）濃縮膠緩沖液貯液（濃縮膠緩沖液貯液（

9、mLmL）分離膠緩沖液貯液（分離膠緩沖液貯液（mLmL）甘油（甘油（mlml）雙蒸水（雙蒸水（mLmL）1010過(guò)硫酸銨（過(guò)硫酸銨（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 水平式電泳裝置水平式電泳裝置Bioinformatics,

10、 2010-2011, Shanxi Agricultural University -3-3二硫二硫 蘇糖醇或蘇糖醇或4 4-5-5Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 低分子量標(biāo)準(zhǔn)蛋白試劑盒低分子量標(biāo)準(zhǔn)蛋白試劑盒: :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 胰蛋白酶抑制劑胰蛋白酶抑制劑 MW=20,100MW=20,100 雞蛋清溶菌酶雞蛋清溶菌酶 MW=14,400MW=14,400Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-2011, Shanxi Agricultural University 將電泳儀的正極與下槽連接，負(fù)極與上槽連接，打?qū)㈦娪緝x的正極與下槽連接，負(fù)極與上槽連接，打開(kāi)電泳儀開(kāi)關(guān)，開(kāi)始時(shí)電流

12、為開(kāi)電泳儀開(kāi)關(guān)，開(kāi)始時(shí)電流為10 mA10 mA，待樣品進(jìn)入分，待樣品進(jìn)入分離膠后，將電流調(diào)至離膠后，將電流調(diào)至20-30 mA20-30 mA，當(dāng)溴酚藍(lán)染料距硅，當(dāng)溴酚藍(lán)染料距硅膠框膠框1cm1cm時(shí)，停止電泳，關(guān)閉電源。時(shí)，停止電泳，關(guān)閉電源。Bioinformatics, 2010-2011, Shanxi Agricultural University Staking gelSeparating gel垂直電泳裝置垂直電泳裝置Bioinformatics, 2010-2011, Shanxi Agricultural University Bioinformatics, 2010-20

13、11, Shanxi Agricultural University 水平式電泳裝置水平式電泳裝置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 固定固定試劑試劑 時(shí)間時(shí)間 500ml500ml乙醇乙醇100ml100ml冰醋酸冰醋酸400ml400ml蒸餾水蒸餾水. .浸泡浸泡75ml75ml乙醇乙醇17g17g醋酸鈉醋酸鈉1.25ml251.25ml25戊二醛戊二醛0.5g0.5g硫代硫酸鈉硫代硫酸鈉.5H.5H2 2O O用用ddddH2O溶解后加至溶解后加至250ml30mins30mins步驟步驟至少至少30mins30mins漂洗漂洗用蒸餾水漂洗用蒸餾水漂洗3 3次次5mins/5mins/次次Bioinformatics, 2010-2011,

15、Shanxi Agricultural University 銀染銀染試劑試劑 時(shí)間時(shí)間 0.25g0.25g硝酸銀硝酸銀50ul50ul甲醛甲醛用用ddHddH2 2O O溶解后溶解后加至加至250ml250ml顯色顯色6.25g6.25g碳酸鈉碳酸鈉25ul25ul甲醛甲醛用用ddH2O加至加至250ml2-10mins2-10mins視蛋白視蛋白帶顯示深棕色帶顯示深棕色步驟步驟20mins20mins終止終止3.65gEDTA-Na3.65gEDTA-Na2 2.2.2H2O用蒸餾水加至用蒸餾水加至250ml250ml10mins10mins漂洗漂洗保存保存用蒸餾水漂洗用蒸餾水漂洗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 涂抹痕跡可能由于多種原因引起，但

17、是最常見(jiàn)的原因是聚丙烯酰胺凝膠倒膠的不均勻或者上樣量過(guò)大。這塊膠倒膠不正確，在上部膠倒入制膠槽之前就已經(jīng)發(fā)生聚合。首先倒入的膠聚合發(fā)生的太快了。與其重新倒膠，不如停止倒膠重新準(zhǔn)備新的聚丙烯酰胺混合液，然后將新的膠倒入舊膠的上方。但是顯然這種連接不是非常牢固。（我推薦重新倒膠，制膠很關(guān)鍵，如果你后續(xù)還要做blot，膠沒(méi)有跑好浪費(fèi)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 條紋拖尾條紋拖尾例例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 部分前端指示劑部分前端指示劑- -例例1 1Bioinformatics, 2010-2011, Shanxi Agricultural University 部分前端指示劑部分前端指示劑- -例例2 2Bioinformatics, 2010-2011, Shanxi Agricultural University 部分前端指示劑部分前端指示劑- -例例3 3Bioinformatics, 2010-2011, Shanxi Agricultural University 癥狀癥狀5-5-條帶僅在凝膠頂部條帶僅在凝膠頂

21、部過(guò)早終止電泳過(guò)早終止電泳例例1 1 Bioinformatics, 2010-2011, Shanxi Agricultural University 過(guò)早終止電泳過(guò)早終止電泳- -例例2 2Bioinformatics, 2010-2011, Shanxi Agricultural University 癥狀癥狀6-6-混雜因素混雜因素扭曲的條帶扭曲的條帶Bioinformatics, 2010-2011, Shanxi Agricultural University 加樣孔不成形加樣孔不成形 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 多種癥狀多種癥狀- -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