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来自 : 蚂蚁淘
ClaireWalczakJanuary1996
YoucantestwhetherornotyouhavegottenanimmuneresponsetothepeptideandhowstrongthatimmuneresponseisbydoingELISAsagainstpeptideconjugatedtoBSA.ByconjugatingtoBSA,youwilleliminateanysignalforantibodiesgeneratedtoKLHduringimmunization.
Protocol:
A.CouplingPeptidetoBSA
Youneed:
- 1mlof2mg/mlBSAin0.1MNaHCO3.
- 1mlof0.2%glutaraldehydein0.1MNaHCO3.
- 0.5mlpeptide(1mg/mlinDMSO).Peptidecanbeaddedasasolidifsoluble.
- Mix,addingglutaraldehydelast.PeptideandBSAturnalittleyellowevenbeforeaddingglutaraldehyde.
- Incubate90"at37degC.
- Add0.1volumesof0.1MNaBH4in0.1MNaHCO3.Therewillbesomebubbling.AddsameamountofNaBH4after15".Doaquickmicrofugespiniftherearetoomanybubbles.
B.ELISAwithPeptideconjugatedtoBSA
- Coat10ug/mlantigen(peptideconjugatedtoBSA)dilutedinTBS(50ul/well)ONat4degC.
- Removeantigenandrinsewells2XswithTBST.
- Block2hrwith200ulof5%NFDMinTBST.
- Removeblockingreagentandrinsewells2XswithTBST.
- IncubateinprimaryantibodydilutedinBlockingBufferfor2hratRT.Idotriplingdilutionsbeginningat1/10(50ul/well).
- Removeprimaryantibodyandrinsewells4XswithTBST.
- Incubateinsecondaryantibody(1/5000Goatanti-rabbitconjugatedtoAP)dilutedinBlockingBufferfor1hratRT(50ul/well).
- Removesecondaryantibodyandrinsewells4XswithTBST.
- Rinsewells2Xswith50mMHCO3;0.5mMMgCl2,pH10.
- Developin1mg/mlp-Nitrophenylphosphatein50mMHCO3;0.5mMMgCl2,pH10(50ul/well).
- ReadA410inELISAreader.
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