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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