小鼠肾损伤分子1(Kim1)Elisa试剂盒_小鼠Elisa试剂盒上海恪敏...
来自 : 蚂蚁淘
CharacteristicsoftheprocedurePreparationofisolatednuclei-procedurePreparationofrADIoactivelabelednucleiMaterial
| Characteristicsoftheprocedure:Pretreatmentofepithelialcellswith20礛CytochalasinBfor30mininRPMIpriortoharvestingresultsinthereductionofcytoskeletalMICROFILamentsandtheeasierreleaseofthenucleifromthesurroundingCytoskeletonduringcelllysis.ForthepreparationofJurkatnuclei,thestepofcytochalasintreatmentcanbeomitted.Inputofabout1E+07cells.Lysisinhypotonicmedium(about23mMcomparedwith137mMinisotonicmedium)usingaDouncehomogenizer(Btypepestle:clearanceabout0.7mm)andpurificationoffreenucleibycentrifugationthroughsucrosesolutionofmediumconcentration(30%=0.88M).Yield:between30%and50%isagoodyield. |
PREPARATIONOFISOLATEDNUCLEI-PROCEDURE- Growcellsina160cm2flask(25mlmedium)uptoabout75%celldensity.
- Removemediumfromthecellcultureflaskexcept5ml.Totheremaining5mladd25祃of4.2mMCytochalasinB(CB);Incubate30minat37癈(cellswillshowanaltereredmorphologyafterwards).
- RemovetheCBcontainingsupernatantandharvestcellsbytrypsinizationandcentrifugationat200gfor10min.
- Washcellstwotimeswith10mlPBS,pH7.2.
- Washcellsoncewith5mlNucleiBuffer(NB).
- ResUSPendcellsin10volumesofNB(including10礛CytochalasinB)(totalvolumeshouldbeatleast1,5mlifyouusea2mlDouncehomogenizer).
- Letcellsswellonicefor20minto30min(controlswellingprocessundermicroscope).
- GentlelysiswithaDouncehomogenizer:About25to50controlled,butdeterminedstrokes,dependingoncelltype;checkliberationofnucleiandgradeofpurityofnucleibyexaminationofsampleunderphasecontrastmicroscope.
- Layerliberatednucleiover30%sucrose(0.88M)inNB:About500祃lysateover2ml30%sucroseina6mlroundbottomedPPcentrifugetube.
- Spinnucleidownat800gfor10min;aspiratesupernatant,thensuckawaythetoplayerandtheinterphase,thoroughly.
- Optionalforhigherpurityofnuclei:takenuclearpelletupin500祃NBandlayernucleiasecondtimeover30%sucrose(0.88M)inNB.Spindownat800gfor10min,andaspiratesupernatant.
- Forawashstep,takenuclearpelletupin3mlNB(atthispointtakeasampleforcountingnucleiinhemcytometerunderphasecontrastmicroscope),andcentrifugeat800gfor10min,aspiratesupernatant.
- Resuspendnucleipelletinnucleistoragebuffer(NSB)at2E+08nuclei/mlor,incaseofradioactivelabellednuclei:resuspendinNSBat1E+07nuclei/mlanddistributethesuspensionin5祃aliqots(50000nuclei).
- Storenucleiat-70癈inaliquotsuntilrequired(uptoseveralweeks).
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PreparationofradioactivelabelednucleiForthepreparationofradioactivenuclei,ALVA31cellswereharvestedand5x106cellswerereseededin162cm2tissuecultureflaskincompleteRPMI+2礐i/ml[3H]-thymidine.After24hofincubation,labeledcellswereharvestedandthenucleiwerepreparedfromthosecellsasdescribedintheprotocolabove.Thedegreeoflabelingwasabout1cpm/nucleus.
| MATERIAL |
| NUCLEIBUFFER(NB): |
| COMPOSITION: | RECIPEfor50ml: |
| 10mMPIPES(pH7.4)10mMKCl2mMMgCl21mMDTT0.1mMPMSF2礸/mlLeupeptin2礸/mlPepstatin2礸/mlAprotinin | 10mlof50mMPIPES,pH7.4500祃of1MKCl1.0mlof100mMMgCl50祃of1MDTT500祃of10mMPMSF100祃of1mg/mlLeupeptin100祃of1mg/mlPepstatin100祃of1mg/mlAprotinin37.65mlH2Onucleasefree |
| NUCLEISTORAGEBUFFER(NSB): |
| COMPOSITION: | RECIPEfor1ml: |
| 10mMPIPES(pH7.4)5mMEGTA80mMKCl20mMNaCl50%Glycerol250mMSucrose1mMDTT0.2mMSpermine0.5mMSpermidine0.1mMPMSF2礸/mlLeupeptin2礸/mlPepstatin2礸/mlAprotinin | 200祃of50mMPIPES(pH7.4),incl.25mMEGTA80祃of1MKCl20祃of1MNaCl500祃Glycerol85mgSucrose1祃of1MDTT10祃of20mMSpermine10祃of50mMSpermidine10祃of10mMPMSF2祃of1mg/mlLeupeptin2祃of1mg/mlPepstatin2祃of1mg/mlAprotinin106祃H2Onucleasefree |
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