Gen5 数据分析软件杭州生科仪器有限公司相关交流-蚂蚁淘在线

Gen5 数据分析软件杭州生科仪器有限公司

蚂蚁淘 /发布时间:1630085406 /浏览量:354

SteveHahn

LastmodifiedFri,Oct16,1998

Beforetryingthismethod,tryrapidproteinprepwhichinvolvesboilingyeastinSDSPAGEbuffer

1.Grow100mlyeasttoA600~1.0.Iftheyeastareovergrown,useproportionallylesscellsfortheprep.2.Collectcellsbycentrifugationandwashin20mlcoldextractionbuffer(containingDTTandproteaseinhibitors).Keepeverythingcoldfromthispointon.3.ResUSPendcellsin600microlitersextractionbufferandtransferto13x100mmglasstesttubesonice.Add400microlitersglassbeads.4.Inthecoldroom,vortexfor1minoniceandkeeponiceforatleast1minwhilevortexingothertubes.5.Repeatthevortexingstep(step4)foratotalof5times.6.Spincellsinprecooledclinicalcentrifugefor3minattopspeed.7.Usinga1mlpipetman,removethesupernatant,leavingmostoftheglassbeadsbehind(transferringsomeoftheyeastisOK).Transfertoamicrocentrifugetube.8.Inthecoldroom,spinattopspeedinthemicrofugefor30min.Removesupernatanttoanewtubebeingcarefulnottocarryoveranycelldebris.9.MeasureproteinconcentrationusingBioRadassayandfreezeinaliquots.Load20-40microgramsonproteingelforwesternanalysis.

ExtractionBuffer:200mMTrispH8.0150mMAmmoniumsulfate10%glycerol1mMEDTAAddfreshondayofextractpreparation:2mMDTTPMSFBenzamidineLeupeptinPepstatinChymostatinProteaseinhibitors0.1MPMSF(100x)16mg/mlEthanolStoreat-20degrees0.2MDTT(100x)32mg/mlH2OStorefrozenat-20degreesBenzamidine(100X)31mg/mlH2O.Storefrozenat-20degreesLeupeptin(500X)0.15mg/mlEthanol.Storeat-70degreesforlessthan6monthsPepstatin(200X)0.28mg/mlmethanolStoreat-20degrees.Chymostatin(2,500X)5mg/mlDMSOStorefrozenat-20degrees

================ 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。