- [05-13]脑出血病人手术后昏迷状态下能不能打白蛋白_寻医问药网_xywy.com
- [10-03]白蛋白的功效与作用
- [10-03]肝功能白蛋白高一点是怎么回事_肝病_
- [10-03]打白蛋白对身体有好处吗
- [09-29]人血白蛋白有什么作用 专家文章 博禾医生
- [09-29]到底癌症病人打白蛋白有何用处？
- [09-29]白蛋白正常值范围是多少_有问必答_
- [10-03]人体白蛋白过高有什么危害?_有问必答_
- [09-29]输白蛋白作用是干什么的_有问必答_
James Hardwick's angiotensin assay protocol
蚂蚁淘
/发布时间:1545758279 /浏览量:56
1.Immunoprecipitatetheproteinfrom3x106cellsusing45microlitersS.aureusbugs.ResUSPendtheimmunoprecipitatesin12microliterskinasebuffer(40mMPIPES,pH7.0,10mMMnCl2)at4°C.2.Thekinasereactioniscarriedoutatroomtemperatureinatotalvolumeof20microlitersthatcontains: 15microCigamma-32P-ATP5microlitersimmunoprecipitate2mMolar[Val5]-angiotensinII 3.Remove5microliteraliquotsofthereactionmixtureafter1,3,and5minutesofreactiontime.TheincorporationofATPislinearduringthisperiod. The5microliteraliquotsthatareremovedateachtimepointaremixedimmediatelywith15microlitersof5%trichloroaceticacid(TCA).Letthesamplessitoniceforatleast15mintoprecipitateproteins.4.Spinthesamplesinamicrocentrifugefor5minat4°C.5.Spot10microlitersofeachsupernatantontoa2x2cmphosphocellulosepaper(Whatmanp81).Washthepaperin0.425%phosphoricacid(add5ml85%phosphoricacidto1LH2Otomake0.425%phosphoricacid.)for3min(15milliliters/pieceofpaper).Repeatthewashtwice.UnincorporatedATPwillbewashedoffthepaper.6.Addthepiecesofpaperto5mlscintillationfluidtoandcount.
================ 蚂蚁淘在线 ================ 免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容 版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。
ThisspecificprocedurewasdevelopedtoassaytheactivityoftheLckkinaseexpressedfromaretroviralvectorinratfibroblasts.Youcanobviouslyusefewerormorecells,dependingonyourneeds.Don"tskimptoomuchontheamountofS.aureusbugsthatyouuseforprecipitationoryouwillrisklosses.Val5-angiotensinIIisobtainedfromSigmaandstoredasa50mMstockat-70°C.Note:Ifyougetmorethan100,000cpmincorporated,youmaybepeggingtheassay.Inthiscase,repeattheexperimentanduselessoftheprecipitateintheassay.ConsumptionofmostoftheATPcanoccurifyouarestudyingproteinsover-expressedbytransienttransfection.
