LEVELIII

Materials

• SUSPensioncultureoffibroblastcells(1liter)
• 35mMTris-HCl,pH7.4,140mMNaCl(TBSbuffer)
• 10mMTris-HCl,pH7.5,10mMKCl,and1.5mMmagnesiumacetate(TBS-M)
• 10XTBS-M
  200mMTris-HCl,pH7.5,1200mMKCl,50mMmagnesiumacetateand70mM-mercaptoethanol
• 10Xsolutionof20aminoacids
• Teflonhomogenizer
• Refrigeratedpreparativecentrifuge
• Sat.(NH)SO
• TBS-Mplus20%(v/v)glycerol
• 1XTBS-Mbuffercontaining0.25Msucrose
• 1XTBS-Mbuffercontaining1.0Msucrose
• SephadexG-25columnequilibratedwith1XTBS-Mbuffer
• Liquidnitrogenstorage
• Reactionmixtureforproteinsynthesis,containingthefollowinginatotalvolumeof50µl2

  Tris-HCl,pH7.5	1.5µm
  Mgacetate	0.15-0.20µm
  KCL	4.0-5.0µm
  -Mercaptoethanol	0.25µm
  ATP	0.05µm
  GTP	0.005µm
  Creatinephosphate	0.50µm
  Creatinekinase	8.0µg
  Eachof19aminoacids(-leucine)	2.0nmol
  C-leucine(150mCi/mmol)	0.125µCi
  Ribosomefraction	1to2Aunits
  ViralmRNAorGlobin9SmRNA	2.0to5.0µg
  or
  PolyU3	10.0µg

  Procedure4

  1. Chillthesuspensionculture(app.10cells)rapidlyinanicebath.Collectthecellsasapellet,bycentrifugationat600xgfor10minutesat4°C.ResuspendthecellsinTBSbufferandwashthemthreetimeswithcoldTBSbuffer.

  2. SuspendthefinalpelletintwovolumesofTBS-Mfor5minutesat0°Candhomogenizethecellswith10to20strokesinatightfittingTeflonhomogenizer.

  3. Foreach0.9mlofhomogenate,add0.1mlofconcentrated10XTBS-Mbuffer.Centrifugethemixtureat10,000xgfor10minutesat4°C.

  4. Decantandcollectthesupernatantextractandadjusttheextractsuchthatthefollowingareaddedtoyieldfinalconcentration:
     ATPto1.0mMATPGTPto0.1mMGTPCreatinephosphateto10mMCreatinekinaseto160µg/mlAminoacidsto40µmeach

  5. Incubatethemixturefor45minutesat37°C.

  6. Centrifugethemixtureat10,000xgfor10minutesatroomtemperature.CoolthesupernatantandpassthesupernatantthroughaSephadexG-25columnat4°C.

  7. TurnonaUVspectrophotometerandadjustthewavelengthto260nm.BlanktheinstrumentwithTBSbuffer.

  8. CentrifugethefiltrateexcludedfromtheSephadexcolumnat165,000xgfor90minutesat4°C.

  9. Precipitatetheproteinswithinthesupernatantbytheadditionofsaturated(NH)SOtoyieldafinal60%(NH)SO.Collecttheprecipitatebycentrifugation.

  10. DissolvetheprecipitateinTBS-Mbufferanddialyzeitagainstthesamebuffercontainingglycerol.

  11. Suspendtheresultingribosomepelletin1XTBS-Mbuffercontaining0.25Msucrose.Place5mlofTBSbufferwith1.0Msucroseintothebottomofacentrifugetubeandlayerthesuspendedribosomesontop.Centrifugeat216,000xgfor2.5hoursat4°C.

  12. WashtheresultingpelletwithTBS-Mbuffer,andresuspenditinthesamebufferwith0.25Msucrose.

  13. DeterminetheribosomeconcentrationusingaUVspectrophotometertomeasuretheA.Theextinctioncoefficientforribosomesis12Aunitspermgpermlat260nm.

  14. Theribosomesmaybefrozenandstoredinliquidnitrogen,orusedforinvitroproteinsynthesis.Iffrozen,theyshouldbethawedonlyoncepriortouse.

      Totestforproteinsynthesis,preparethereactionmixtureforproteinsyntheis.

  15. Incubatethereactionmixtureat37°Cfor60minutes.Terminatethereactionbypipetting40µlofthemixtureontoa2.5cmdiskofWhatman3MMfilterpaper.Dipthediskintocold10%TCAfor15minutesandthenin5%TCAat90°Cfor15minutes.

  16. Rinsethedisktwicein5%TCAfor5minutes,onceinalcohol:ether(1:1),andthendryit.

  17. Placethediskintoascintillationvialandaddatoluene-basedfluor.RefertoAppendixHfordetailsontheuseofascintillationcounter.

  18. MeasuretheamountofrADIoactivelylabeledaminoacidincorporatedintoprotein.

  19. Graphtheproteinsynthesizedversustime.

  Optional

  Foradvancedwork,comparetheactivityofribosomesisolatedfromthefibroblastculturestothoseisolatedfromaprokaryoteculture,aplant(yeastorpeaseedlings)andfromgeneticµtantsknowntoalterthestructureofeitherrRNAoranyoftheribosomestructuralproteins.

  IfsourcesofmRNAareavailable(orifthetimeistakentoextractRNAandelectrophoreticallypurifyit),thencomparisonsoftherateofproteinsynthesiscanbemadewithinthissystem.Finally,oncetheproteinsaresynthesized,theycanbeseparatedonSDS-PAGEandratesofsynthesiscanbedeterminedbybothtimeofincorporationofleucineandbyincreasingmolecularweights.ThelatterwillalsodemonstratethepossIBLepresenceofµltipleRNA'swiththesystem,whereastheradioactivitywillmaskthepresenceofheterogenousRNA.

  ================  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。