CoIP 冷泉港protocolWord下载.docx
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CoIP 冷泉港protocolWord下载.docx
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Dimethylpimelimidate(DMP)(dimethylpimelimidatedihydrochloride;
e.g.,Aldrich360341)Ethanolamine(200mM,pH8.0)
Glycine(100mM,pH3.0)
IPbuffer
M9saltsolution
NaN3(sodiumazide)(optional;
seeStep28)
Nematodegrowthmedia(NGM)plates,6-cm,containgstarvedworms
Nematodegrowthmedia(NGM)plates,10-cmand14.5-cm
OP50solution
ProteinA-agarose(Roche)
ReagentsforBradfordassay
RNaseA(10mg/mL;
e.g.,adilutionofRNaseA[100mg/mL]fromQiagen19101)(optional;
seeStep32)
SDSsamplebuffer(2X)(preheatedforStep31.i)
Equipment
Adaptersfor15-mLcentrifugetubes
Centrifugetubes(15-mLglass;
e.g.,Kimble45500)
EquipmentforSDS-PAGEandWesternblotanalysis
Falcontubes(15-mLand50-mL)
Filter(0.45-µ
m)
Gloves(protective;
canbethickwintergloves)
High-speedcentrifuge(e.g.,BeckmannCoultercentrifugeAvantiJ-20orJ-25)
Ice
Laminarflowhood
Liquidnitrogen
Liquid-nitrogencontainers(two)
Microcentrifugetubes(1.5-mL)
Micropipetteandtips(including1-mLtips)
Mortarandpestle(prechilled)
Rotorforcentrifuging15-mLglasstubes(e.g.,JA25.50)
Sieve(metal)
Spatula(metal,prechilled)
Spoon(metal)
Syringe(10-mL)
Tabletopcentrifugefor15-mLFalcontubes(e.g.,Eppendorfcentrifuge5702)
Tabletopmicrocentrifuge
Testtuberotationwheelfor1.5-mLtubes
Waterbathpresetto96°
C
METHOD
Large-ScaleGrowthofC.elegans
Thissection(Steps1-11)describestheculturingofalargenumberofmixed-stagewormsunderphysiologicalconditions.Toavoidanaccumulationofinhibitorypheromonesandthusadelayinwormdevelopmentoraninductionofstarvation,atwo-stepplate-basedamplificationprotocolisused.Wormsarefrozeninliquidnitrogenintheformofpearlstofacilitateusageofsmallerportions(insteadoftheentirewormculture)forextractpreparation(Step12).
1.Take1210-cmNGMplatesand3014.5-cmNGMplates,andseedeachofthemwith1mLor2mLofOP50solution,respectively.Drytheplatesunderalaminarflowhoodandstoreovernightatroomtemperature.Keepplatesat4°
Cforlong-termstorage.
2.Preparethewormsfromstarved-wormplatesasfollows:
i.Takethree6-cmstarved-wormplatesandwashthewormsoffthreetimeswith1mLofM9saltsolutioninto1.5-mLtubes.
ii.Pelletthewormsbycentrifugationfor1minat400g.
iii.Putthewormsonicefor2mintosettlethem.Removethesupernatant.
iv.Washthewormsthreetimeswith1mLofM9saltsolution.
3.Resuspendthewormsin1.2mLofM9saltsolution.Plate100µ
Lofresuspendedwormsoneachofthe1210-cmNGMplatesunderalaminarflowhood,andlettheplatesdryfor5minwiththelidsopen.
4.Letthewormsgrowat20°
Cfor4duntilthefoodisalmostconsumed.
5.Feedthewormsbyadding1mLofOP50solutionontotheplatesunderthelaminarflowhood,andlettheplatesdrywiththelidsopen.
6.Incubatethewormsat20°
Cuntilthefoodisalmostconsumed(usuallyonemoretimeovernight).
7.Treatthewormsasfollows:
i.WashthewormsofftheplateswithM9saltsolutionandcollectthemin15-mLFalcontubes.ii.Storethewormsonice.
iii.Pelletthewormsbycentrifuginginatabletopcentrifugeat600gfor2min.
iv.WashthewormpelletthreetimeswithM9saltsolution.
8.Resuspendthewormsin60mLofOP50solutionanddistribute2mLper14.5-cmNGMplate(30platesintotal).Drytheplatesunderthelaminarflowhoodandincubatethewormsat20°
CuntiltheyreachatleasttheL4stage.
Avoidstarvingtheanimals!
9.Treatthewormsasfollows:
i.HarvestthewormsbywashingthemofftheplateswithM9saltsolutionandcollectthemin15-mLFalcontubesonice.
ii.WashthewormsthreetimeswithM9saltsolution.
iii.WashthewormstwiceinbufferB70supplementedwithproteaseinhibitors.
10.Collectallwormsinoneortwo15-mlFalcontubes(notmorethan4.5mLofsettledwormspertube)andaddanequalvolumeofbufferB70supplementedwithproteaseinhibitors.Theyieldofsettledwormsis~3-6mLandisexpandedtoatotalvolumeof6-12mLaftertheadditionofbuffer.
Anequivalentof~2mLofsettledwormsisplentyforatypicalIPexperimentwithtwoindividualsamples.
11.Freezethewormsasfollows:
i.Resuspendthewormswellandletthemdripintoacontainerofliquidnitrogenbygentlypushingthemoutofa1-mLmicropipettetip.
ii.Collectthefrozenwormpearlsuspensionwithasieveplacedoveranemptyliquid-nitrogencontainer.
iii.Transferthewormpearlstoa50-mLFalcontubeusingametalspoon.Atthispoint,thewormpearlscanbestoredat–80°
C.
Whole-WormExtractPreparation
Thissection(Steps12-15)describesanefficientandeconomicalprotocolfortheproductionofcytoplasmicextractfromfrozenwormculturesunderconditionsthatpreservetheintegrityofcellularproteinsandRNA.Performtheentireprocedureat4°
Cunlessotherwisestated.
12.Useanequivalentof2mLofsettledwormsforanIPexperimentwithtwoindividualsamples,ormoredependingonthenumberofIPsamplesintheexperiment.Grindthewormpearlstoafinepowderinliquidnitrogenusingamortarandpestlethathavebeenprecooledwithliquidnitrogen.Maintainthewormhomogenateasacoldpasteduringtheentiregrindingprocedurebyaddingfreshliquidnitrogentothemortaronceithasevaporated.Grindthewormsseveraltimestoproduceafinepowder.
Becarefulwhentouchingthecooledmortar.Itisnecessarytowearprotectiveglovestopreventcoldburns.
13.Transferthepowderinto15-mLglasstubeswithaprecooledspatulaandadd2mLofbufferB70(supplementedwithfreshproteaseinhibitors).
Thepowderproducedfromanequivalentof2mLofsettledwormsfillsapproximatelyhalfthevolumeofthe15-mLglasstube.
14.Centrifugeasfollows:
i.Centrifugethetubesinahigh-speedcentrifugeat37,000gfor30minat4°
ii.Transferthesupernatanttoprecooled1.5-mLtubes.
iii.Centrifugeagaininatabletopcentrifugeat20,000gfor10minat4°
15.Passthesupernatantthrougha0.45-µ
mfilterintoaprecooledFalcontubeusinga10-mLsyringe.Proceedimmediatelytoco-IP(Step29).KeepsomeextractasidetodeterminetheproteinconcentrationinaBradfordassayandasinputcontrolsforimmunoblots.
Theconcentrationsmayvarybetween10and35mgofproteinpermilliterofextract.CovalentCouplingofAntibodiestoResin
16.Placeabedvolumeof15µ
LofProteinA-agaroseperIPsampleinto1.5-mLtubes.Settlethebeadsbycentrifugationinatabletopcentrifugeat400gfor30sec.
Dependingontheantibodysource,ProteinG-agarosemightbepreferred(seeReagents).
17.Washthebeadsthreetimeswith500µ
LofIPbuffer.
18.Resuspendthebeadsinatotalvolumeof500µ
LofIPbufferincludingtheantibody(usually30µ
Lofserumor40µ
gofpurifiedantibody,whichvariesdependingontheaffinityandconcentrationoftheantibody).
19.Rotateonawheelfor1hatroomtemperatureorovernightat4°
20.Washthebeadsoncein500µ
Lofboratebuffer.
21.Resuspendthebeadsin300µ
22.Add200µ
Lofboratebuffercontaing0.0026gofDMP.
23.Rotateonawheelfor30minatroomtemperature.
24.Washthebeadswith1mLofethanolamine.
25.Incubatethebeadsin500µ
Lofethanolaminefor2h,rotatingonawheelatroomtemperature.
26.Washthebeadsquicklyin500µ
Lof100mMglycine,pH3.0.
27.Washthebeadsoncein500µ
28.WashthebeadstwiceinbufferB70andstoreat4°
Cuntiluse.Ifthebeadsaretobestoredforlongerthanaday,add0.02%NaN3.
Co-IPfromWormExtracts
29.Preparea40-µ
LbedvolumeofProteinA-agarosebywashingthreetimesinbufferB70.Add
1.1mLoffreshextractandincubatefor30minonawheeltoprecleartheextractofmoleculesthatnonspecificallybindtoProteinA-agarose.
ThisissufficientmaterialforoneIPexperimentconsistingoftwosamples:
aprotein-specificantibodyandanonspecificcontrolantibody.
30.Pelletthebeadsfor30secat400gandusethepreclearedsupernatantfortheIP.
31.Removeaninputsamplefromthepreclearedextract:
i.Add30µ
Lofpreclearedextractto30µ
Lofhot2XSDSsamplebuffer.
ii.Incubatefor2minat96°
iii.Storeat–20°
32.(Optional)TreatwithRNaseAasfollows:
i.Add10µ
Lof10mg/mLRNaseAto1mLofpreclearedextract,androtatethemixturefor15minonawheel.
ii.Removea50-µ
LsamplefromthepreclearedextractbeforeandaftertheRNaseAtreatment.iii.PurifytotalRNAfromthesamples.
iv.AnalyzetheefficiencyoftheRNaseAdigestonanagarosegel.ProceedtoStep33.
33.Add500µ
Lofextracttoeachsampleofantibody-coupledbeadsandincubatefor2honawheel.
34.WashthebeadsquicklythreetimesinbufferB70,thenwashtwiceinbufferB70for10minonawheel.
35.Removethesupernatantuntiltheliquidlevelbarelytouchesthebeads.
36.Elutetheproteinsfromthebeadsasfollows:
i.Add20µ
Lof2XSDSsa
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