重组蛋白的纯度分析ppt下载_PPT免费下载
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Purpose
TheprotocoldescribeshowtopreparehumantonsillysateforuseinpurificationofICAM-1,LFA-1,andPNAd.
Materials
- SafetyEquipmentsLabCoatLatexGlovesFaceMaskBenchPaper
- Freshhumantonsiltissue
- Parafilm
- Scissors
- Stainlesssteelmeshscreen(200mesh=74mm,Type316;Tylinter,Mentor,Ohio)
- RPMI-1640medium
- TritonX-100
- LysisBuffer:50mMTris-HCl,pH7.5150mMNaCl0.02%NaN310mg/mlAprotinin10mg/mlPepstatin10mg/mlLeupeptin1mMPMSF(Madefreshthedayofexperiment.Dissolvein100%EtOH)1mMBenzamidine5mMIodoacetamide
- WhatmanPaper
- CentrifugeBottles
- Funnel
- Squeezebottle
- SeveralBeakers
- Homogenizer(FisherPowerGen125)
- 50mlCorningtubes
- IceBucket&Ice
- Douncehomogenizer(40mlPyrexCatNo.7727-40)
- 1literbottle
- Stirbar
- 2Side-armflasks
Procedure
Allworkmustbedoneat4oC.- Collect50gramsoftonsiltissue.(thisshouldyieldalargeamountofICAM-1,LFA-1,andPNAd)
Note1:Collectingthismuchtonsilscantakesometime.Soitisgoodtocollectfrommorethanonesource.
Note2:Youcanprepeachsampleasyoureceiveitorwaituntilyouhavea~50goftonsils.
- Ifyoudecidetoprepthemrightawaythenperformsteps2to4andthenfreezeat-80oC.
- Ifyouwanttowaituntilyouhave~50goftonsil,youshouldimmediatelyfreezetonsilsat-80oC.
- Clearaworkareainthe4oCwalk-infridge.Laydownbenchpaperandassembleallyoumaterials.
- Mincethetonsilswiththescissors.TryandmakethepiecesasmallaspossIBLe.
Note:Youcanuseasmallglassplateasacuttingsurfacetomakethejobeasier.
- Foldthewiremeshintheshapeofaconeandplaceinafunnel.Placefunnelonthemouthofthebeaker,tocollectRPMI-1640.
- PourRPMI-1640intoasqueezebottle.WashthemincestonsilswithcopiousamountsofRPMI-1640.Washasmallamountoftonsilsatatime.
(Atthispointyoucanfreezethetonsilsuntilyouhaveenough)
- Setupthehomogenizerwiththelargeprobe(1cmdiameter).
- Putasmallamountofmincedtonsilinthe50mlCorningtubeandaddlysisbufferto~10mlmark.
- Insertprobetothetubeandthecovertheneckofthetubeandthemainbodyoftheprobewithparafilm.Thisisveryimportant.Thispreventsaerosolsfromescapingduringthehomogenizingprocess.
- Homogenizeinsmallbursttoavoidheatingthesample.
Note1:Homogenizetillyouliquefythesample.Youmaystillseealittlebitofwhitestrands,buttheseareconnectivetissueandwillnotcompletelybreakdown.
Note2:Theprobewillgetcloggedwithconnectivetissue.Cleanitofwithtissuepaperandrinseinwithwaterbypulsingtheprobetoremovetherestofthestuckconnectivetissue.Oncecleanagainyoucancontinuehomogenizingyoursample.
- Collectthehomogenizedtissueinabeakerthatiskeptonice.
- Usingthedouncehomogenizer,homogenizethecollectedsampleusinganup&downandleft&righthandmotion.Dothisatleast10timestogetaveryfinesample.
Note:Toavoidspillingyousample,don’tusemorethanthevolumestatedonthesideofthedouncehomogenizer.
- Whenyouaredonetransferhomogenizedtissuetoa1literbottleandaddinthestirbar.
- Bringthevolumeupto1literbyaddingmoreoftheLysisbuffer.
- AddTriton-X100toafinalconcentrationof2%byvolume.
Note:TheTriton-X-100willnotdissolveimmediately.Itwillgointosolutionslowlyovernight.
- Stirgentlyovernightinthe4oCwalk-in.
- Transfertheliquidtocentrifugebottlesandspindownlysateat8000gfor1hour.
- Decantlysateinacleanbeaker.Discardpellet.
- Filterlysatetwice,usingBucknerFunnelandWhatmanpaperasfollows:
- CuttheWhatmanpapertocoverthebottomoftheBucknerfunnel.
- Placefunnelonside-armflaskandattach.
- Attachthefirstflasktothesecondandtothevacuum.
- FilterthelysatethroughtheWhatman,changingitoftenasitgetsclogged.
- Filtratemaynowbestoreda4oCuntilitistimetorunoveranaffinitycolumn.
Note:Don"tstoreformorethanacoupleofdaysat4oC.
References
K.D.Puri,E.B.Finger,G.Gaudernack,andT.A.Springer(1995).SialomucinCD34isthemajorL-selectinligandinhumantonsilhighendothelialvenules.JCellBiol131:261-270.
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