大豆蛋白提取.docx

3.8.ProteinExtractionTotalproteinsfromdifferentsamples(0.3g)wereextractedin1mLcoldTPBSbuffer(150mMNaCl,8mMNa2HPO4·12H2O,2mMKH2PO4·H2O,4mMKCl,0.05%Tween-20,and2%SDS,pH7.4).Aftershakingfor30minutesatroomtemperature,themixturewascentrifugedtwiceat20,000ginarotor(modelAvantiJ-25,Beckman)for15mineach.TotalproteinconcentrationwasdeterminedbyBCA(Bicinchoninicacid)assay(MultisciencesBiotechnologyCo.Ltd.,Hangzhou,China),usingbovineserumalbumin(BSA)asastandard.3.9.WesternBlottingAnalysisThepredictedproteinproductofCP4-EPSPScomprises527aminoacidswithmolecularweightof55.5kD,whichincludesa72aminoacidchloroplastictransitpeptideprovenpreviously[13],indicatingamatureproteinof47.6kDfollowingthecleavagesiteofthetransitpeptide.Inourexperiment,onepeptidesequencecontainingthespecificaminoacidscorrespondingtothematureCP4-EPSPSsequencepositions(S19→R33)fortheantigenwereobtainedbychemicalsynthesis.Thepeptidewashydrophilic,surface-oriented,andflexible.SyntheticpeptideswerepurifiedbyHPLCandcoupledtokeyholelimpethaemacyanin(KLH).TheCP4-EPSPS-KLHswerecollectedandusedforproducingtherabbitpolyclonalantibody(SC-16).Forthewesternblotanalysis,protein(30μg)orproteinextractedfromanequalweightofdifferentsampleswassubjectedtoSDS-PAGEusinga12.5%acrylamideresolvinggel(MiniProteanIISystem,Bio-Rad,Hertz,UK).Separatedproteinswerethentransferredtopolyvinylidenedifluoride(PVDF)membranes,andnon-specificbindingofantibodieswasblockedwith5%non-fatdriedmilkinphosphate-bufferedsaline(PBS,pH7.4)overnightat4°C.Membraneswerethenincubatedovernightat4°C,withprimaryantibodiesdiluted1:3000inPBSbufferplus1%non-fatdriedmilk.Immunecomplexesweredetectedusinghorseradishperoxidase(HRP)-conjugatedgoatanti-rabbitimmunoglobulinG.Thecolorwasdevelopedwithasolutioncontaining3,3'-diaminobenzidinetetrahydrochloride(DAB)astheHRPsubstrate.Meanwhile,identicalSDS-PAGEgelwasstainedbyCoomassieBrilliantBlue.