protocolsWord文件下载.docx
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protocolsWord文件下载.docx
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1.3.3.T-cellsfromspleen
1.3.4.FibroblastcelllineNIH3T3
1.4.Human
1.4.1.Serum
1.4.2.Toothpulptissue
2.IEF(Isoelectricfocusing)
2.1.In-gelrehydration
2.2.Samplecuploading
3.SDS-PAGE(SDS-polyacrylamidegelelectrophoresis)
3.1.CastingtheSDSgels
3.1.1.2-DSDS-PAGE
3.1.2.1-DSDS-PAGE
3.2.Electrophoresis
3.2.1.2-DSDS-PAGE
3.2.2.1-DSDS-PAGE
4.Proteindetection
4.1.Silverstaining
4.2.WesternBlotting
5.Proteinidentification(massspectrometry)
6.Troubleshooting
7.Linkstootherprotocols
8.References
9.Chemicals
APS
Bis
CHAPS
DTT
PMSF
PonceauS
SDS
TCEP
TEMED
Tris
AmmoniumPersulfate
N,N`-Methylene-bisacrylamide
3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate
1,4-dithio-DL-threitol
PhenylmethanesulfonylFluoride
3-Hydroxy-4-(2-sulfo-4-[4-sulfophenylazo]phenylazo)-2,7-naphthalenedisulfonicacidsodiumsalt
Sodiumdodecylsulfate
Tris-(2-carboxyethyl)phosphinehydrochloride
N,N,N´
N´
-Tetramethylethylenediamine
Tris-[hydroxymethyl]aminomethane
TheE.colistrainRB791wasgrowninLBmediumandcells(50-100ml)collectedatdifferentgrowthstages.Afteracentrifugation(10min,8000rpm,4°
C)thecellswerewashedtwicewith2mlTE-PMSF(1mMEDTA,0.1MTris,14µ
MPMSF,pH7.5).Thepelletwasthenresuspendedin3mlTE-PMSF,disruptedbyFrenchPresswith900PSI(62.1bar)andthecelllysatecentrifuged(30min,15.300rpm,4°
C).Thesupernatantwascollectedandtheproteinconcentrationdeterminedwithacommerciallyavailablekit(RotiNanoquant)accordingtoBradford,1976.Aliquotsof100µ
gweredried(SpeedVac)andstoredat-20°
C.
Priortoseparationinthefirstdimension(IEF)theproteinswereresuspendedin350µ
lureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris/HCl,pH7.5,1mMEDTA,14µ
MPMSF)andtransferedbyin-gelrehydrationintotheIEFgels.
FortheanalysisofthefermentativegrowththeS.cerevisiaewildtypestrainBJ1991wascultivatedonrichYPD(1%(w/v)yeastextract,2%(w/v)peptone,and2%(w/v)D-glucose)andcollectedatanopticaldensity(600nm)of1.1.ForthestudyoftherespirationawildtypeculturewasgrowninYPDtoanopticaldensity(600nm)of1-2,thenpelleted,andtransferredtoYPGcontaining3%(v/v)glycerolinsteadofD-glucose.After16hcellswerecollected,centrifuged(10min,8000rpm,4°
C)andwashedtwicewith2mlTE-PMSF(1mMEDTA,0.1MTris,14µ
MPMSF,pH7.5).Thenthepelletwasresuspendedin3mlTE-PMSFanddisruptedbyFrenchPresswith900PSI(62.1bar).Thecelllysatewascentrifugated(30min,15.300rpm,4°
C)andthesupernatantcollected.Theproteinconcentrationwasdeterminedwithacommerciallyavailablekit(RotiNanoquant)accordingtoBradford,1976.Aliquotsof100µ
Yeastcellsweregrownandcollectedasdescribedabove.ThemitochondriawereisolatedaccordingtoMeisingeretal.,2000.Inbrief,cellswerepelletedbycentrifugation(3.000g,5min),washedwithdistilledwaterandresuspendedunderslowlyshakingfor20minat30°
Cin2ml/gDTTbuffer(100mMTris-H2SO4pH9.4,10mMDTT).Afterwashingwithzymolyasebuffer(1.2Msorbitol;
20mMpotassiumphosphate,pH7.4)cellswereincubatedwith5mg/g(w/v)Zymolyase-20T(SeikagakuKogyoCo.)in7ml/gzymolyasebufferfor45minat30°
Cforconversationintospheroblasts.Homogenizationwascarriedoutby15strokesinaglass-Teflonpotterin6.5ml/gice-coldhomogenisationbuffer(0.6Msorbitol;
10mMTris-HCl,pH7.4;
1mMEDTA,1mMPMSF;
0.2%(w/v)bovineserumalbumin).Thishomogenatewasdilutedwith1volice-coldhomogenisationbufferandcelldebrisandnucleiremovedbycentrifugation(1500g,5min,4°
C).Thesupernatantwasagaincentrifuged(3000g,5min,4°
C)andthemitochondriainthesupernatantpelletedbyadditionalcentrifugation(12.000g,15min,4°
C).ThepelletwaswashedwithSEM(250mMsucrose;
1mMEDTA;
10mMMOPS,pH7.2),againcentrifuged(12.000g,15min,4°
C)andresuspendedinSEMtogiveafinalconcentrationof5mg/ml.Themitochondrialfractionwastreatedby10strokesinaglass-Teflonpotterandloadedontoathree-stepsucrosegradient(1.5ml60%,4ml32%,1.5ml23%,1.5ml15%(w/v)sucroseinEMbuffer(10mMMOPS,pH7.2;
1mMEDTA).Aftercentrifugation(134.000g,1h,2°
C)thepurifiedmitochondriawererecoveredfromthe60%/32%interface,dilutedwith2volSEMandcentrifuged(10.000g,2°
C).Themitochondrialpelletwasresuspendedinureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris-HCl,pH7.5,1mMEDTA,14µ
MPMSF)andtheproteinconcentrationdetermined.Proteinaliquots(100µ
g)werestoredat-20°
Priortoseparationinthefirstdimension(IEF)thevolumeoftheureabufferwasadjustedto350µ
landtheproteinstransferedbyin-gelrehydrationintotheIEFgels.
Kidneysofadultmice(14.5days)weredissected,washedin5volofice-cold20mMTris/HClpH7.8,10mMEDTA,2mMEGTA,2mMDTT,dried(SpeedVac)andresuspendedinureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris-HCl,pH7.5,1mMEDTA,14µ
MPMSF).Theproteinconcentrationwasdeterminedandaliquotsof100µ
gstoredat-20°
Bones(calvariaandfemur)from3-4weekoldmicewerecollected,freezedwithliquidnitrogenandgrindedwithapestletoafinepowder.Proteinswereprecipitatedwithacetone(80%acetone,0.12%(w/v)DTT)foratleast2hat–20°
C,pelletedbycentrifugation(30min,14.000rpm,4°
C)andresuspendedinureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris-HCl,pH7.5,1mMEDTA,14µ
Priortoseparationinthefirstdimension(IEF)theIEFgelswereincubatedin350µ
lureabufferwithoutprotein.PriorseparationtheproteinsolutionwasappliedintosamplecupsplacedattheanodicendoftheIEFgel.
Spleentissuewashomogenized,lymphocytes(B-andT-cells)wereisolatedusingalymphoprep-gradientcentrifugation.ThepurifiedlymphocyteswereaddedtoaTcellpurificationcolumnandtheT-cellscollectedandcentrifuged.Thepelletwasresuspendedinureabuffer(7Murea,2Mthiourea,2%(w/v)CHAPS,1%(w/v)DTT,2%(v/v)carrierampholytes3-10,100mMTris-HCl,pH7.5,1mMEDTA,14µ
MPMSF)accordingtoGö
rgetal.,2000.Theproteinconcentrationwasdeterminedandaliquotsof100µ
C.
Fortheseparationinthefirstdimension(IEF)theIEFgelswereincubatedin350µ
lrehydrationsolution(6Murea,2Mthiourea,1%(w/v)CHAPS,0.4%(w/v)DTT,0.5%(v/v)carrierampholytes3-10)accordingtoGö
rgetal.,2000withoutprotein.Priortoseparationinthefirstdimension(IEF)theproteinsolutionwasappliedintosamplecupsplacedattheanodicendoftheIEFgel.
Acultureofamousefibroblastderivedcellline(NIH3T3)wasgrownfor48hinDulbecco`smodifiedEaglemedium(DMEM)containing3%fetalcalfserum(FCS).CellswererinsedoncewithDMEMwithoutFCSandcollectedfromtheplates(10cm)bycarefullyscratching.Proteinswereprecipitatedwithacetone(80%acetone,0.12%(w/v)DTT)foratleast2hat–20°
C)andresuspendedinureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris-HCl,pH7.5,1mMEDTA,14µ
Fortheseparationinthefirstdimension(IEF)thevolumeoftheureabufferwasadjustedto350µ
Theserumwascollected,proteinscentrifugeddownandwashedin1mlTE-PMSF(1mMEDTA,0.1MTris,14µ
MPMSF,pH7.5).Thentheproteinpelletwasprecipitatedinacetone(80%(v/v)acetone,0.12%(w/v)DTT)foratleast2hat–20°
C)andresuspendedinureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10).Theproteinconcentrationwasdeterminedandaliquotsof100µ
Intactandcarious(advancedenamelorearlytomoderatedentinalcaries)thirdmolarswereremoved.Externalsurfacesoftheteethwerefirstcleanedwith75%(v/v)ethanola
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