海拉细胞,你一定要知道
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Phycoerythrinconjugationprotocol
David"smethodmodifiedfromreferences(2)and(3).IusedthismethodtoconjugateamouseIgG2amonoclonalantibody(IB4).Itworkswell.
| Ab | Antibody,about2.5mg/ml |
| PE | R-Phycoerythrin,purified(MolecularprobesP-801in60%ammoniumsulfate) |
| PBS | PhosphatebufferedsalinewithoutCa2+/Mg2+ |
| MeOH | Methanol |
| DMSO | Dimethylsulphoxide,anhydrous |
| SPDP | 3-(2-pyridyldithio)propionicacidN-hydroxysuccinimideester(Sigma) |
| SMCC | Succinimidyltrans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate(MolecularProbes) |
| DTT | Dithiothreitol |
| NEM | N-ethylmaleimide |
| * | 10mlSephadexG-10columns(2) |
| Separationcolumn | AlongSephacrylS-300column.Iused117x1cm.Youcan"treallygetawaywithanythingsmaller.Usealongthinoneratherthanashortfatone,sincethelatterdidnotworkforme.Separationoftheconjugatefromthefreereagentsistricky. |
| * | Centricon30andCentriprep30centrifugalconcentrators(Amicon) |
| * | Roomtemperatureisabout22°C |
1.PreparationofPE
- R-PEfromMolecularProbes.PartnumberP-801,R-PEin60%NH4SO4at4mg/ml.0.25mlofP-801.Spin10minsonlowinmicrofuge.Discardsupernatant.ResUSPendPEin0.25mlPBS.Dialyzetwicefor30minsversus150mlPBSatroomtemperature.
2.SPDPmodificationofPE
- AdjustPEtoapprox1.4mg/ml(=0.7ml)inPBSAdd16µlSPDP(1.3mg/mlinMeOH)Incubateatroomtemperaturefor2.5hours
- ***Youmusttimesteps3and4tofinishatthesametime,sincethereactionproductsareunstable***
3.SMCCmodificationofantibody
- 1mlpurifiedantibodyatapprox2.5mg/mlinPBS.Antibodymustbefairlypure-i.e.purifyonproteinAorGcolumnbeforeconjugating.Add20µlSMCC,1.7mg/mlinDMSO.Incubatefor1hour.
4.DTTtreatmentofSPDP-PE
- Takeproductfromstep(2)andadd30µlDTT(0.5M,i.e.77mg/mlinPBS).Incubatefor30minsatroomtemperature.
5.Purificationofreactants
- SeparatederivatizedPEandAbfromactivatorsonG-10columns.Youshouldnowhaveapproximately2mlofeachreactant.
6.Conjugation
- Mixderivatizedreagentsandincubateat4°Covernightonrotarymixer.
7.Stopreaction
- Add80µlN-ethylmaleimide(0.1mM)toreactionmixturetostopreaction.Agitateandincubate30minsatroomtemperature
8.Concentrateproduct
- Concentrateto0.5mlorlessonCentriconconcentrator(Centricon30or100arefine)
9.Separateconjugate
- Applytotopofseparationcolumn.ElutewithPBS.Pumpcolumnatapproximately0.3ml/min.Orderofelutionisconjugatefirst(MW~400,000)thenfreePE(MW~240,000)thenfreeAb(MW~150,000).Begincollecting1.0mlfractionswhenpinkbandapproachesbottomofcolumnandcontinuefor10fractionsafterithasdisappeared.
10.Evaluateseparation
- MeasureOD280andOD565-theformerisprotein,thelatterthePEabsorptionpeak.Testactivityofeachfractionagainstknownreactivecellsbyflowcytometry.Plotalltheseresultsonagraphandestimatewherethemostconjugateis(Seefigurebelow).Poolthesefractions.
11.Finalconcentration
- ConcentratepooledconjugatefractionsusingCentriprepandCentriconconcentrators.Add0.5%sodiumazide.Storeat4°Cprotectedfromlight.Donotfreeze.
Figure1.AnalysisofcolumneluatefractionsfromPEconjugationofmonoclonalantibodyIB4(murineIgG2a,anti-humanCD18).Theopticaldensityofeachfractionwasmeasuredat280nmand565nm(OD280andOD565respectively).AnaliquotofeachfractionwasusedtostainhumanneutrophilswhichwerethenanalyzedusingaFACScanflowcytometer(Becton-Dickinson).MediancellularfluorescenceintensityintheFL2(orangefluorescence)channelisshown.
References
- ChambersJD,SimonSI,BergerEM,SklarLA,ArforsK-E.Endocytosisofbeta2integrinsbystimulatedhumanneutrophilsanalyzedbyflowcytometry.JournalofLeukocyteBIOLOGy53:462-469(1993)
- KronickMN,GrossmanPD.Immunoassaytechniqueswithfluorescentphycobiliproteinconjugates.Clinicalchemistry29:1582-1586(1983)
- OiVT,GlazerAN,StryerL.Fluorescentphycobiliproteinconjugatesforanalysesofcellsandmolecules.JournalofCellBiology93:981-986(1982)
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