蛋白酶活性染色方法

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1、2.4.SDS–PAGEandzymogramanalysisSDS–PAGEwasperformedona5%stackinganda12%runninggelaccordingtothemethodofLaemmli(1970),andproteinbandswerevisualizedbystainingwithCoomassieBrilliantBlueG250(Sigma).ZymographywasperformedonSDS–PAGEasdescribedbyHadderetal.(2009).Afterelectro

2、phoresis,thegelwasincu-batedat40Cfor1hin50mMTris–HCl(pH8.0)containing1%casein.Theactivebandwasvisualizedbystainingwith1%CoomassieBrilliantBlueG250anddestainingwithmethanol–glacialaceticacid–water(30:10:60).1.2.3目的蛋白的电泳洗脱纯化按Laemmli[5]的方法,用SDS-PAGE首先将TB24kDa蛋白进行分离,分离胶浓度

3、为12.5%,浓缩胶浓度为4%,厚度1.5mm,采用自制的大孔疏子(见图1),点样量分别为40、140、160μl。电泳完毕后,切下胶板中两条40μl条带在考马斯亮蓝中染色,以作对照,剩余胶块置20%三氯乙酸中固定30min,将已染色的胶块和未染色的胶块对齐,切下未染色胶块中相应的TB24kDa蛋白带,将其捣碎成1mm3碎片,放入透析袋中,加入少量新鲜电泳缓冲液。把透析袋放入装有SDS-PAGE电泳缓冲液的水平电泳槽中,有胶块的一端靠近负极,30mA电泳4-5h,然后倒转电极电泳5min。电泳结束后,经聚乙二醇(PEG-20000)

4、浓缩,吸取透析袋中的浓缩液,再用浓缩管CentrexUF-2,12,000rPmin,浓缩20min。最后,11,000rPmin,4℃离心15min,除去沉淀的SDS,收集上清液,再经SDS-AGE检验洗脱结果。2.3.5.Detectionofproteolyticactivityonpolyacrylamidegels(proteasezymogram)参考“Twodetergentstablealkalineserine-proteasesfromBacillusmojavensisA21:Purification,chara

5、cterizationandpotentialapplicationasalaundrydetergentadditive”ZymographywasperformedinconjunctionwithSDS–PAGEaccordingtothemethoddescribedbyGarcia-Carrenoetal.(1993)withslightmodification.Thesamplewasnotheatedbeforeelectrophoresis.Afterelectrophoresis,thegelwassubmerged

6、in100mMglycine–NaOHbuffer(pH10.0)containing2.5%TritonX-100for30min,withconstantagitationtoremoveSDS.TritonX-100wasthenremovedbywashingthegelthreetimeswith100mMglycine–NaOHbuffer(pH10.0).Thegelwasthenincubatedwith1%(w/v)caseinin100mMglycine–NaOHbuffer(pH10.0)for20minat5

7、0℃.Finally,thegelwasstainedwithCoomassieBrilliantBlueR-250.Thedevelopmentofclearzonesonthebluebackgroundofthegelindicatedthepresenceofproteaseactivity.2.6.Casein-SDS-PAGECasein-SDS-PAGEwasperformedasdescribedbyHummeletal.(1996)withafewmodifications.Thesampleswereelectro

8、phoresedat4℃ina12.5%gelcontaining0.2%casein.Samplesweremixedwithloadingbuffer(withoutb-mercaptoethanolordithioreitol)a