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.

Phycoerythrin&nbsp;conjugation&nbsp;protocol

Phycoerythrinconjugationprotocol

1.PreparationofPE

2.SPDPmodificationofPE

3.SMCCmodificationofantibody

4.DTTtreatmentofSPDP-PE

5.Purificationofreactants

6.Conjugation

7.Stopreaction

8.Concentrateproduct

9.Separateconjugate

10.Evaluateseparation

11.Finalconcentration

References

Phycoerythrin conjugation protocol