《生化分析检测技术》PPT课件

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1、2.1.1吸光光度法AbsorbanceAssay(280nm)ConsiderationsforuseAbsorbanceassaysarefastandconvenient,sincenoadditionalreagentsorincubationsarerequired.Noproteinstandardneedbeprepared.Theassaydoesnotconsumetheprotein.Therelationshipofabsorbancetoproteinconcentrationislinear.Becausediffere

2、ntproteinsandnucleicacidshavewidelyvaryingabsorptioncharacteristicstheremaybeconsiderableerror,especiallyforunknownsorproteinmixtures.Anynon-proteincomponentofthesolutionthatabsorbsultravioletlightwillinteferewiththeassay.Cellandtissuefractionationsamplesoftencontaininsoluble

3、orcoloredcomponentsthatinterfere.Themostcommonusefortheabsorbanceassayistomonitorfractionsfromchromatographycolumns,oranytimeaquickestimationisneededanderrorinproteinconcentrationisnotaconcern.Anabsorbanceassayisrecommendedforcalibratingbovineserumalbuminorotherpureproteinsol

4、utionsforuseasstandardsinothermethods.Absorbanceassays1PrincipleProteinsinsolutionabsorbultravioletlightwithabsorbancemaximaat280and200nm.Aminoacidswitharomaticringsaretheprimaryreasonfortheabsorbancepeakat280nm.Peptidebondsareprimarilyresponsibleforthepeakat200nm.Secondary,t

5、ertiary,andquaternarystructureallaffectabsorbance,thereforefactorssuchaspH,ionicstrength,etc.canaltertheabsorbancespectrum.2EquipmentInadditiontostandardliquidhandlingsuppliesaspectrophotometerwithUVlampandquartzcuvettearerequired.3AnalysisUnknownproteinsorproteinmixtures.Use

6、thefollowingformulatoroughlyestimateproteinconcentration.Pathlengthformostspectrometersis1cm.Concentration(mg/ml)=Absorbanceat280nmdividedbypathlength(cm.)Pureproteinofknownabsorbancecoefficient.Usethefollowingformulaforapathlengthof1cm.Concentrationisinmg/ml,%,ormolaritydepe

7、ndingonwhichtypecoefficientisused.concentration=Absorbanceat280nmdividedbyabsorbancecoefficientToconvertunits,usetheserelationships:Mgprotein/ml=%proteindividedby10=molaritydividedbyproteinmolecularweightUnknownswithpossiblenucleicacidcontamination.Usethefollowingformulatoest

8、imateproteinconcentration:Concentration(mg/ml)=(1.55xA280)–(0.76xA26