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分光光度计知识
Withtheaidofspectroscopy,thequantitativeanalysisofnucleicacidsandproteinshasestablisheditselfasaroutinemethodinmanylaboratories.Itincludesabsorptionmeasurementsintheultravioletandinthevisibilityrange.Proteinsaremeasured(directly)at280nm,nucleicacidsat260nmandcolorimetricproteindeterminationiscarriedoutintherangefrom550to600nm.TheBioPhotometeroffersthefollowingpre-installedtestprocedures:
1.NucleicaciddeterminationDNA,RNA,oligonucleotidesandevenmononucleotidescanbemeasureddirectlyinaqueoussolutionsinadilutedorundilutedform.Aqueousbufferswithlowionconcentrations(e.g.TEbuffer)areidealforthismethod.Theconcentrationisdeterminedbymeasuringat260nmagainstblankandthenevaluatingviafactor.Normally,theuserhastocalculatetheconcentrationofthemeasuredsampleusingaccordingfactors.Incontrast,theBioPhotometercanchangethesefactorseasilyandwilldoallnecessarycalculations.
Theabsorptionof1OD(A)isequivalenttoapproximately50µg/mldsDNA,approximately33µg/mlssDNA,40µg/mlRNAorapproximately30µg/mlforoligonucleotides.PuritydeterminationofDNAInterferencebycontaminantscanberecognizedbythecalculationof“ratio”.TheratioA260/A280isusedtoestimatethepurityofnucleicacid,sinceproteinsabsorbat280nm.PureDNAshouldhavearatioofapproximately1.8,whereaspureRNAshouldgiveavalueofapproximately2.0.Absorptionat230nmreflectscontaminationofthesamplebysubstancessuchascarbohydrates,peptides,phenolsoraromaticcompounds.Inthecaseofpuresamples,theratioA260/A230shouldbeapproximately2.2.
2.ProteindeterminationTheproteincontentofapreparationcanbedeterminedonthebasisofvariousdifferentanalyticalprocedures.Evaluationcanbecarriedoutviafactororviaacalibrationcurve,withuptotenstandardsintheBioPhotometer.
Absorptionmeasurementat280nm(A280)A280methodmaybeusedwithinconcentrationsofuptoapproximately4mg/ml(3.0A).Thismethodissimpleandrapid,butmaybedisturbedbytheparallelabsorptionofnon-proteins(e.g.DNA).Unlikethecolorimetricprocess,thismethodislesssensitiveandrequireshigherproteinconcentrationsandshouldthusbeusedwithpureproteinsolutions.Inadditiontothedirectabsorbancedisplay,evaluationispossIBLewiththeBioPhotometerviatheWarburgformulaorviastandard.
Colorimetricdetermination(dyetests)Proteinsamplesoftenconsistofacomplexmixtureofmanydifferentproteins.Thequantitativedetectionoftheproteincontentisusuallyachievedonthebasisofthereactionsshownbyfunctionalgroupsoftheproteinstodye-formingreagents.Theintensityofthedyecorrelatesdirectlywiththeconcentrationofthereactinggroupsandcanbemeasuredexactly.
Lowryassay595nmSpecialistliteraturecontainsamultitudeofmodificationsfortheLowryassay.Theprincipaltargetistoreducethehighsusceptibilitytointerference.IncomparisontothepureBiuretassay,thesensitivityofthisassayhasgreatlyincreased.However,theLowrymethodisadverselyaffectedbyawiderangeofnon-proteins.AdditivessuchasEDTA,ammoniasulfateorTritonX-100inparticularareincompatiblewiththetest.
Bicinchoninineacidassay562nm(BCA)ThistestrepresentsahighlyregardedalternativetotheLowryassay.Itiseasiertocarryoutandsensitivitycanbevariedusingdifferenttemperatures.FurThermore,thedyecomplexisverystable.However,thistestishighlysusceptibletointerference,althoughonthepositiveside,itsinsensitivitytodetergentsissimilartothatoftheLowrymethod.
Bradfordassay595nmThismethodistwiceassensitiveastheLowryorBCAtestandisthusthemostsensitivequantitativedyeassay.Itistheeasiesttohandleandmostrapidmethodandhastheadditionaladvantagethataseriesofreducingsubstances(e.g.DTTandmercaptoethanol),whichinterferewiththeLowryorBCAtest,havenoadverseeffectonresults.However,itissensitivetodetergents.Themaindisadvantageisthatidenticalamountsofdifferentstandardproteinscancauseconsiderabledifferencesintheresultingabsorptioncoefficients.
3.BacterialcelldensityThedensityofbacterialsUSPensionsmaybemeasuredphotometricallyat595nmwithoutdyeshavingtobeadded.Thisappliese.g.tothepreparationofcompetentcells(i.e.cells,whichareabletoabsorbplasmidDNA),thatmustbeinaspecificphaseofgrowth.
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