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MessagePosté le: Ven 28 Oct - 23:06 (2016)    Sujet du message: Current Protocols In Protein Science Pdf Download Répondre en citant




Current Protocols In Protein Science Pdf Download > bit.ly/2eNELOt











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Anti-HA,,,protein,,,should,,,only,,,detect,,,the,,,full-length,,,uncleaved,,,protein,,,in,,,the,,,lysate,,,exposed,,,to,,,BILN-2061.,,,External,,,links[edit].,,,Immediately,move,the,plate,to,the,hood,,then,quickly,dispense,into,the,center,of,each,well,200,μL,of,the,2x,reducing,SDS,lysis,buffer.,(See,,“Critical,,Parameters.”)2.,,Subclone,,each,,complete,,TimeSTAMP,,cassette,,in,,frame,,to,,either,,the,,N-,,or,,C-terminus,,of,,the,,gene.The,,complete,,cassette,,consists,,of,,N-terminal,,T7,,epitope,,tag,,,N-terminal,,cleavage,,site,,,NS3,,protease,,,C-terminal,,cleavage,,site,,,and,,C-terminal,,HA,,tag.,,Alternatively,,,,add,,,1,,,μL,,,of,,,25U/μL,,,benzonase,,,nuclease,,,and,,,Mg2+,,,to,,,1,,,mM,,,final,,,concentration,,,to,,,each,,,sample,,,,mix,,,well,,,,and,,,incubate,,,at,,,room,,,temperature,,,for,,,30,,,minutes.Sonication,,,is,,,faster,,,but,,,requires,,,the,,,availability,,,of,,,a,,,sonicator,,,and,,,familiarity,,,with,,,its,,,use.,,,After,lysis,buffer,has,been,dispensed,into,all,the,wells,,agitate,the,plate,to,spread,the,lysis,buffer,around,the,entire,surface,of,the,wells.The,purpose,of,the,wash,step,is,to,remove,serum,components,,primarily,albumin,,whose,presence,in,the,lysate,causes,anomalous,migration,during,electrophoresis,and,ghosting,during,immunoblotting.,After,accomplishing,this,for,all,wells,,quickly,aspirate,away,the,wash,buffer.,When,adding,BILN-2061,at,later,time,points,,it,is,best,to,make,a,30μM,solution,of,BILN-2061,in,media,to,be,used,as,a,10x,mix,instead,of,adding,BILN-2061,directly,from,the,DMSO,stock,onto,the,cells.,

For,proteins,bearing,the,TimeSTAMP,cassette,at,the,N-terminus,,the,roles,of,the,T7,and,HA,epitopes,are,reversed.,For,,,each,,,construct,,,being,,,tested,,,,prewarm,,,two,,,tubes,,,each,,,of,,,1,,,mL,,,of,,,standard,,,culture,,,media,,,with,,,serum.,,,Tsien,,ude.dscuneistr,,tsienlab.ucsd.edu)BILN-2061,(Boehringer,Ingelheim),,10-30mM,in,DMSOreagents,for,cloning,and,PCRcell,type,of,interestcell,culture,mediatransfection,reagent2x,SDS,lysis,buffer2-mercaptoethanolPBS,or,HBSSsonicator,with,microtip,,or,25U/μL,benzonase,nuclease,(Novagen)SDS-PAGE,and,immunoblotting,equipment,and,reagentsrat,2F2,(Roche),or,mouse,12CA5,(Roche),or,mouse,HA-7,(Sigma),or,chicken,anti-HA,(ICL),antibodymouse,anti-T7,antibody,(Novagen)secondary,antibodies,for,detection,by,chemiluminescence,or,fluorescence,chemiluminescence,substrate,(if,performing,chemiluminescence)autoradiography,film,(for,chemiluminescence),or,immunoblot,imaging,system,(for,chemiluminescence,and,fluorescence).,To,,,study,,,protein,,,production,,,rates,,,over,,,a,,,defined,,,time,,,window,,,with,,,treatments,,,of,,,interest,,,,repeat,,,steps,,,6-7,,,above,,,,calculating,,,how,,,many,,,wells,,,are,,,needed,,,for,,,all,,,experimental,,,conditions,,,(which,,,may,,,be,,,a,,,combination,,,of,,,treatments,,,to,,,be,,,tested,,,and,,,time,,,windows,,,in,,,which,,,protein,,,synthesis,,,will,,,be,,,observed).For,,,instance,,,,if,,,cells,,,are,,,to,,,be,,,either,,,untreated,,,and,,,treated,,,with,,,a,,,growth,,,factor,,,and,,,protein,,,synthesis,,,over,,,two,,,different,,,time,,,windows,,,analyzed,,,,then,,,at,,,least,,,seven,,,wells,,,are,,,necessary:,,,two,,,time,,,points,,,with,,,BILN-2061,,,multiplied,,,by,,,two,,,treatment,,,conditions,,,,plus,,,one,,,well,,,as,,,a,,,transfected,,,but,,,untreated,,,and,,,uninhibited,,,control,,,,a,,,transfected,,,well,,,that,,,is,,,treated,,,continuously,,,with,,,BILN-2061,,,,and,,,an,,,untransfected,,,well.16.,,,For,,each,,construct,,to,,be,,tested,,,transfect,,the,,two,,allocated,,wells,,with,,0.8,,μg,,DNA,,each,,with,,a,,liposomal,,reagent,,or,,with,,calcium,,phosphate.Perform,,liposomal,,or,,calcium,,phosphate,,transfections,,according,,to,,standard,,protocols,,,except,,use,,only,,0.8,,μg,,of,,DNA,,to,,avoid,,toxicity,,,adjusting,,all,,other,,volumes,,in,,the,,transfection,,mixture,,accordingly.8.,,It,,also,,provides,,a,,quantitative,,method,,for,,measuring,,rates,,of,,protein,,synthesis,,from,,a,,reporter,,construct,,within,,a,,defined,,time,,window,,under,,different,,stimulation,,or,,treatment,,conditions.,,References[edit].,,Basic,Protocol:,Testing,and,use,of,TimeSTAMP,tags,by,immunoblotting.,Verify,,proper,,functioning,,of,,the,,TimeSTAMP,,cassette,,6.,,

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