细胞生物学：蛋白质定位与功能研究方法探讨.pptxVIP

1ThinkingandTesting(12-03-2008)1.Youradvisorisexplaininghislatestresultsinyourweeklylabmeeting.ByfusinghisproteinofinteresttoGFP,hehasshownthatitislocatedentirelyinthenucleus.Buthewondersifitisatruenuclearproteinorashuttlingproteinthatjustspendsmostofitstimeinthenucleus.Heisunsurehowtoresolvethisissue.HeknowsthatleptomycinB(细霉素B)willnotbehelpfulinthisinstanceandasksforinput.Havingjustreadanarticleabouthowasimilarproblemwasanswered,yousuggestthathemakeaheterocaryonbyfusingcellsthatareexpressinghistaggedproteinwithanexcessofcellsthatarenotexpressingit.Youtellhimthatinthepresenceofaproteinsynthesisinhibitortoblocknewsynthesisofthetaggedproteinhecanresolvetheissuebyexaminingfusedcellswithtwonuclei.Hegivesyouapuzzledlookandaskshowdoesthathelp.Youtellhimwhathehassooftentoldyou“Thinkaboutit.”◆句子的理解◆知识点训练◆研究方法◆科学创意◆人文精神◆场景设计◆研究路线◆训练提出问题的能力

2?研究策略路线基因融合基因转化蛋白表达蛋白定位细霉素B抑制蛋白定位细胞A细胞B细胞B细胞培养短暂培养融合异核体细胞新蛋白质合成抑制核质蛋白分析

3问题与答案(12-03-2008)A．Howwouldexaminingthetwonucleiinaheterocaryonanswerthequestion?Whatresultswouldyouexpectiftheproteinwereatruenuclearprotein?Whatwouldyouexpectifitwereashuttlingprotein?B．Whydidyousuggestthataproteinsynthesisinhibitorwouldbeneededinthisexperiment?Ans:A.Inaheterocaryonwithtwonuclei,oneexpressingtheGFP-taggedproteinandtheothernot,itispossibletodecidewhethertheproteinisatruenuclearproteinorashuttlingprotein.Ifitisanuclearprotein,theGFPshouldremainassociatedwithasinglenucleus.Ifitisashuttlingprotein,theGFPwillredistributetobothnuclei.B.ItiscriticalinthisexperimenttoblocksynthesisofnewGFP-taggedprotein.Ifnewproteinweremade,itwouldenterbothnuclei,regardlessofwhetheritwasanuclearproteinorashuttling