資源描述:
《《生化分析檢測(cè)技術(shù)》PPT課件》由會(huì)員上傳分享,免費(fèi)在線閱讀,更多相關(guān)內(nèi)容在教育資源-天天文庫(kù)。
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