生化分析检测技术课件

2.1.1吸光光度法AbsorbanceAssay(280nm)ConsiderationsforuseAbsorbanceassaysarefastandconvenient,sincenoadditionalreagentsorincubationsarerequired.Noproteinstandardneedbeprepared.Theassaydoesnotconsumetheprotein.Therelationshipofabsorbancetoproteinconcentrationislinear.Becausedifferentproteinsandnucleicacidshavewidelyvaryingabsorptioncharacteristicstheremaybeconsiderableerror,especiallyforunknownsorproteinmixtures.Anynon-proteincomponentofthesolutionthatabsorbsultravioletlightwillinteferewiththeassay.Cellandtissuefractionationsamplesoftencontaininsolubleorcoloredcomponentsthatinterfere.Themostcommonusefortheabsorbanceassayistomonitorfractionsfromchromatographycolumns,oranytimeaquickestimationisneededanderrorinproteinconcentrationisnotaconcern.Anabsorbanceassayisrecommendedforcalibratingbovineserumalbuminorotherpureproteinsolutionsforuseasstandardsinothermethods.

Absorbanceassays

1PrincipleProteinsinsolutionabsorbultravioletlightwithabsorbancemaximaat280and200nm.Aminoacidswitharomaticringsaretheprimaryreasonfortheabsorbancepeakat280nm.Peptidebondsareprimarilyresponsibleforthepeakat200nm.Secondary,tertiary,andquaternarystructureallaffectabsorbance,thereforefactorssuchaspH,ionicstrength,etc.canaltertheabsorbancespectrum.2EquipmentInadditiontostandardliquidhandlingsuppliesaspectrophotometerwithUVlampandquartzcuvettearerequired.3AnalysisUnknownproteinsorproteinmixtures.Usethefollowingformulatoroughlyestimateproteinconcentration.Pathlengthformostspectrometersis1cm.Concentration(mg/ml)=Absorbanceat280nmdividedbypathlength(cm.)Pureproteinofknownabsorbancecoefficient.Usethefollowingformulaforapathlengthof1cm.Concentrationisinmg/ml,%,ormolaritydependingonwhichtypecoefficientisused.concentration=Absorbanceat280nmdividedbyabsorbancecoefficientToconvertunits,usetheserelationships:Mgprotein/ml=%proteindividedby10=molaritydividedbyproteinmolecularweightUnknownswithpossiblenucleicacidcontamination.Usethefollowingformulatoestimateproteinconcentration:Concentration(mg/ml)=(1.55xA280)–(0.76xA260)4CommentsColdsolutionscanfogupthecuvette,whilewarmsolutionscanreleasebubblesandinterferewiththereadings.Forconcentratedsolutions(absorbancegreaterthan2)simplydilutethesolution.Absorbancecoefficientsofsomecommonproteinstandards:Bovineserumalbumin(BSA):63Bovine,human,orrabbitIgG:138Chickenovalbumin:70ReferencesLayne,E.SpectrophotometricandTurbidimetricMethodsforMeasuringProteins.MethodsinEnzymology3:447-455.1957.Stoscheck,CM.QuantitationofProtein.MethodsinEnzymology182:50-69.1990.5AbsorbanceAssay(205nm)ConsiderationsforuseSeeconsiderationslistedundertheabsorbanceassayat280nm.Thismethodisjustasconvenientasforabsorbanceat280nm.Itmaybepreferredifthereisexcessivecontaminationbynucleicacids,sincenucleicacidsabsorbverylittleradiationat205nm.Settingthewavelengthisabittrickysince205nmisrightontheshoulderoftheproteinpeak.6Theproblemofanaccuratewavelengthsettingcanbeavoidedbydeterminingabsorbanceat210nm(extinctioncoefficientsrangefrom20to24).Howeverthereislesssensitivityandmorevariationwithbufferconditions.ReferencesScopes,RK.AnalyticalBiochemistry59:277.1974.Stoscheck,CM.QuantitationofProtein.MethodsinEnzymology182:50-69.1990.9DeterminationoftheExtinctionCoefficientforaProteinofUnknownConcentrationConsiderationsforuseTheconcentrationcanbedeterminedforasolutionofapureproteinwithunknownextinctioncoefficient.10EquipmentInadditiontostandardliquidhandlingsuppliesaspectrophotometerwithUVlampandquartzcuvettearerequired.ProcedureDilutethesolutionabout30foldforthereadingat205nmandinclude0.01%Brij35inthebuffertopreventadsorptionofproteinontoplasticorglasssurfaces.11AnalysisUsethefollowingformulatodeterminetheextinctioncoefficientat205nm:E(205nm)=27+120x(A280dividedbyA205)Thereadingat205nmmustbemultipliedbythedilutionfactorbeforeusingtheformula.Next,determineproteinconcentration:Proteinconcentration(M)=A205dividedbyE(205nm)Youcannowdeterminetheextinctioncoefficientfor280nm:E(280nm)=concentration(M)dividedbyA280

12CommentsAnabnormalphenylalaninecontentwillthrowofftheresultconsiderably.Theaccuracyofthetechniquedependsonanaverageaminoacidcomposition.ReferencesScopes,RK.AnalyticalBiochemistry59:277.1974.Stoscheck,CM.QuantitationofProtein.MethodsinEnzymology182:50-69.1990.13Hartree-LowryandModifiedLowryProteinAssays

ConsiderationsforuseTheLowryassay(1951)isanoften-citedgeneraluseproteinassay.Forsometimeitwasthemethodofchoiceforaccurateproteindeterminationforcellfractions,chromatographyfractions,enzymepreparations,andsoon.Thebicinchoninicacid(BCA)assayisbasedonthesameprincpleandcanbedoneinonestep,thereforeithasbeensuggested(Stoscheck,1990)thatthe2-stepLowrymethodisoutdated.However,themodifiedLowryisdoneentirelyatroomtemperature.TheHartreeversionoftheLowryassay,amorerecentmodificationthatusesfewerreagents,improvesthesensitivitywithsomeproteins,islesslikelytobeincompatiblewithsomesaltsolutions,providesamorelinearresponse,andislesslikelytobecomesaturated.TheHartree-Lowryassaywillbedescribedfirst.Colorimetricassays:

14PrincipleUnderalkalineconditionsthedivalentcopperionformsacomplexwithpeptidebondsinwhichitisreducedtoamonovalention.Monovalentcopperionandtheradicalgroupsoftyrosine,tryptophan,andcysteinereactwithFolinreagenttoproduceanunstableproductthatbecomesreducedtomolybdenum/tungstenblue.15EquipmentInadditiontostandardliquidhandlingsuppliesaspectrophotometerwithinfraredlampandfilterisrequired.Glassorpolystyrene(cheap)cuvettesmaybeused.16Procedure-Hartree-LowryassayReagentsReagentAconsistsof2gmsodiumpotassiumtartratex4H20,100gmsodiumcarbonate,500ml1NNaOH,H20tooneliter(thatis,7mMNa-Ktartrate,0.81Msodiumcarbonate,0.5NNaOHfinalconcentration).Keeps2to3months.ReagentBconsistsof2gm2gmsodiumpotassiumtartratex4H20,1gmcoppersulfate(CuSO4x5H20),90mlH20,10ml1NNaOH(finalconcentrations70mMNa-Ktartrate,40mMcoppersulfate).Keeps2to3months.ReagentCconsistsof1volFolin-Ciocalteaureagentdilutedwith15volswater.17AssayPrepareaseriesofdilutionsof0.3mg/mlbovineserumalbumininthesamebuffercontainingtheunknowns,togiveconcentrationsof30to150micrograms/ml(0.03to0.15mg/ml).Add1.0mleachdilutionofstandard,protein-containingunknown,orbuffer(forthereference)to0.90mlreagentAinseparatetesttubesandmix.Incubatethetubes10minina50degreesCbath,thencooltoroomtemperature.Add0.1mlreagentBtoeachtube,mix,incubate10minatroomtemperature.Rapidlyadd3mlreagentCtoeachtube,mix,incubate10mininthe50degreebath,andcooltoroomtemperature.Finalassayvolumeis5ml.Measureabsorbanceat650nmin1cmcuvettes.18AnalysisPrepareastandardcurveofabsorbanceversusmicrogramsprotein(orviceversa),anddetermineamountsfromthecurve.Determineconcentrationsoforiginalsamplesfromtheamountprotein,volume/sample,anddilutionfactor,ifany.19Procedure-modifiedLowry(roomtemperature)ReagentsDissolve20gmsodiumcarbonatein260mlwater,0.4gmcupricsulfate(5xhydrated)in20mlwater,and0.2gmsodiumpotassiumtartratein20mlwater.Mixallthreesolutionstopreparethecopperreagent.Prepare100mlofa1%solution(1gm/100ml)ofsodiumdodecylsulfate(SDS).Preparea1MsolutionofNaOH(4gm/100ml).Forthe2xLowryconcentratemix3partscopperreagentwith1partSDSand1partNaOH.Solutionisstablefor2-3weeks.Warmthesolutionto37degreesCifawhiteprecipitateforms,anddiscardifthereisablackprecipitate.Better,keepthethreestocksolutions,andmixjustbeforeuse.Prepare0.2NFolinreagentbymixing10ml2NFolinreagentwith90mlwater.Keptinanamberbottle,thedilutionisstableforseveralmonths.20AssayDilutesamplestoanestimated0.025-0.25mg/mlwithbuffer.Iftheconcentrationcan'tbeestimateditisadvisabletopreparearangeof2-3dilutionsspanninganorderofmagnitude.Prepare400microliterseachdilution.Duplicateortriplicatesamplesarerecommended.Prepareareferenceof400microlitersbuffer.Preparestandardsfrom0.25mg/mlbovineserumalbuminbyadding40-400microlitersto13x100mmtubes+buffertobringvolumeto400microliters/tube.Add400microlitersof2xLowryconcentrate,mixthoroughly,incubateatroomtemp.10min.Add200microliters0.2NFolinreagentveryquickly,andvorteximmediately.Completemixingofthereagentmustbeaccomplishedquicklytoavoiddecompositionofthereagentbeforeitreactswithprotein.Incubatefor30min.moreatroomtemperature.Useglassorpolystyrenecuvettestoreadtheabsorbancesat750nm.Iftheabsorbancesaretoohigh,theymaybereadat500nn.21CommentsRecordingofabsorbancesneedonlybedonewithin10min.ofeachotherforthismodifiedprocedure,whereastheoriginalLowryrequiredprecisetimingofreadingsduetocolorinstability.Thismodificationislesssensitivetointerferingagentsandismoresensitivetoproteinthantheoriginal.Aswithmostassays,theLowrycanbescaledupforlargercuvettesizes,howevermoreproteinisconsumed.Proteinswithanabnormallyhighorlowpercentageoftyrosine,tryptophan,orcysteineresidueswillgivehighorlowerrors,respectively.ReferencesLowry,OH,NJRosbrough,ALFarr,andRJRandall.J.Biol.Chem.193:265.1951.Oostra,GM,NSMathewson,andGNCatravas.Anal.Biochem.89:31.1978.Stoscheck,CM.QuantitationofProtein.MethodsinEnzymology182:50-69(1990).Hartree,EF.AnalBiochem48:422-427(1972).22BiuretProteinAssay

ConsiderationsforuseTheprincipleofthebiuretassayissimilartothatoftheLowry,howeveritinvolvesasingleincubationof20min.Thereareveryfewinterferingagents(ammoniumsaltsbeingonesuchagent),andLayne(1957)reportedfewerdeviationsthanwiththeLowryorultravioletabsorptionmethods.However,thebiuretassayconsumesmuchmorematerial.Thebiuretisagoodgeneralproteinassayforbatchesofmaterialforwhichyieldisnotaproblem.TheBradfordassayisfasterandmoresensitive.23PrincipleUnderalkalineconditionssubstancescontainingtwoormorepeptidebondsformapurplecomplexwithcoppersaltsinthereagent.EquipmentInadditiontostandardliquidhandlingsuppliesavisiblelightspectrophotometerisneeded,withmaximumtransmissionintheregionof450nm.Glassorpolystyrene(cheap)cuvettesmaybeused.24ProcedureReagentAformulaforbiuretreagentis2.25gmSodiumpotassiumtartrate(f.w.282.22),0.75gmCoppersulfatex5H2O(f.w.249.68),1.25gmPotassiumiodide(166.0),alldissolvedinorderin100ml0.2MNaOH(f.w.40.0).Bringvolumeto250mlwithdistilledwater(scalevolumeupordownasneeded,ofcourse).Discardifablackprecipitateforms.25AssayVolumessample,reagentcanbescaledup/downand/orvolumeratiosvaried,aswithanyassay.Warmupthespectrophotometer15min.beforeuse.Preparestandardsfrombovineserumalbumin,preferablycalibratedusingabsorbanceat280nmandtheextinctioncoefficient.Thereportedsensitiverange,using9mlreagentto1mlsample,isfrom1to10mgprotein.Theactualsensitiverangemayextendbeyondtheupperlimit.Prepareareferencetubewith1mlbuffer.Ifpossible,diluteunknownstoanestimated1to10mg/mlwithbuffer;arangeofdilutionsshouldbeusediftheactualconcentrationcannotbeestimated.Use1mlsampleperassaytubeAdd9mlBiuretreagenttoeachtube,vorteximmediately,andletstand20min.Readat550nm.26AnalysisPrepareastandardcurveofabsorbanceversusmicrogramsprotein(orviceversa),anddetermineamountsfromthecurve.Determineconcentrationsoforiginalsamplesfromtheamountprotein,volume/sample,anddilutionfactor,ifany.27CommentsThecolorisstable,butallreadingsshouldbetakenwithin10min.ofeachother.Aswithmostassays,theBiuretcanbescaleddownforsmallercuvettesizes,consuminglessprotein.Proteinswithanabnormallyhighorlowpercentageofaminoacidswitharomaticsidegroupswillgivehighorlowreadings,respectively.ReferencesGornall,AG,CSBardawill,andMMDavid.J.Biol.Chem.177:751.1949.Layne,E.SpectrophotometricandTurbidimetricMethodsforMeasuringProteins.MethodsinEnzymology10:447-455.1957.Robinson,HWandCGHogden.J.Biol.Chem.135:707.1940.Slater,RJ(ed.).ExperimentsinMolecularBiology.Clifton,NewJersey:HumanaPress,1986.P.269.Weichselbaum,TE.Am.J.Clin.Pathol.Suppl.10:40.194628

布拉德福德蛋白检测

使用注意事项

布拉德福德蛋白检测法非常迅速，而且和劳里法用几乎等量的蛋白质样品.此法相当精确，对于超出范围的样品可以在几分钟内重新测试。此法适合一般分析，特别适用于确定的细胞蛋白质含量以及凝胶电泳蛋白的含量分析。29Assaymaterialsincludingcolorreagent,proteinstandard,andinstructionbookletareavailablefromBio-RadCorporation.Themethoddescribedbelowisfora100µlsamplevolumeusing5mlcolorreagent.Itissensitivetoabout5to200microgramsprotein,dependingonthedyequality.Inassaysusing5mlcolorreagentpreparedinlab,thesensitiverangeiscloserto5to100µgprotein.Scaledownthevolumeforthe"microassayprocedure,"whichuses1mlcuvettes.Protocols,includinguseofmicrotiterplatesaredescribedintheflyerthatcomeswiththeBio-Radkit.30PrincipleTheassayisbasedontheobservationthattheabsorbancemaximumforanacidicsolutionofCoomassieBrilliantBlueG-250shiftsfrom465nmto595nmwhenbindingtoproteinoccurs.Bothhydrophobicandionicinteractionsstabilizetheanionicformofthedye,causingavisiblecolorchange.Theassayisusefulsincetheextinctioncoefficientofadye-albumincomplexsolutionisconstantovera10-foldconcentrationrange.31EquipmentInadditiontostandardliquidhandlingsuppliesavisiblelightspectrophotometerisneeded,withmaximumtransmissionintheregionof595nm,ontheborderofthevisiblespectrum(nospeciallamporfilterusuallyneeded).Glassorpolystyrene(cheap)cuvettesmaybeused,howeverthecolorreagentstainsboth.Disposablecuvettesarerecommended.32ProcedureReagentsBradfordreagent:Dissolve100mgCoomassieBrilliantBlueG-250in50ml95%ethanol,add100ml85%(w/v)phosphoricacid.Diluteto1literwhenthedyehascompletelydissolved,andfilterthroughWhatman#1paperjustbeforeuse.(Optional)1MNaOH(tobeusedifsamplesarenotreadilysolubleinthecolorreagent).TheBradfordreagentshouldbealightbrownincolor.Filtrationmayhavetoberepeatedtoridthereagentofbluecomponents.TheBio-Radconcentrateisexpensive,butthelotsofdyeusedhaveapparentlybeenscreenedformaximumeffectiveness."Homemade"reagentworksquitewellbutisusuallynotassen