QuantitativeDeterminationofPeptidesbySulfhydryl(-SH)GroupsNew(ContributedbyDavidVanHorn,Dept.ofChemistry,UCBerkeleyGregBulaj,Dept.ofBIOLOGy,UniversityofUtah)Thisisthestandard“Ellman’sTest”forthedeterminationoffreethiols.Itworkswellforsmallpeptidesandproteinssynthesizedusingstandardsolidphasesyntheticmethods.LowryProteinAssay(P.J.HansenLab,Dept.ofAnimalSciences,UniversityofFlorida)TheLowryprocedureisoneofthemostvenerableandwidely-usedproteinassays,beingfirstdescribedin1951[Lowryetal.,J.Biol.Chem.193:265-275(1951)].Underalkalineconditions,coppercomplexeswithprotein.Whenfolinphenolreagent(phospho-molyBDic-phosphotungsticreagent)isadded,theFolin-phenolreagentbindstotheprotein.Boundreagentisslowlyreducedandchangescolorfromyellowtoblue.LowryProteinAssayLowryAssayforProteinConcentration(Folin-CiocalteuAssay)LowryProteinAssay(WilliamH.Heidcamp)BradfordProteinAssay(P.J.HansenLab,Dept.ofAnimalSciences,UniversityofFlorida)TheBradfordproteinassayisasimpleprocedurefordeterminationofproteinconcentrationsinsolutionsthatdependsuponthechangeinabsorbanceinCoomassieBlueG-250uponbindingofprotein.Unlikemanyotherassays,includingtheLowryprocedure,theBradfordassayisnotsusceptIBLetointerferencebyawideusr/localietyofchemicalspresentinsamples.Thenotableexceptionishighconcentrationsofdetergents.Thereissignificantprotein-to-proteinusr/localiationinabsorbancevaluesobtainedwiththeBradfordprocedureanditisadvisabletochooseaproteinstandardthatislikelytogiveabsorbancevaluesclosetothosefortheproteinsamplesofinterest.Theassayhereisdesignedforuseinmicrotiterplates.Thisisaneasyassayformatforthosewithaccesstomultiplechannelpipettorsandmicrotiterplatespectrophotometers.BradfordProteinAssay(WilliamH.Heidcamp)BioradProteinAssay:BradfordReagent(SupryaJayadev)BiuretProteinAssay(WilliamH.Heidcamp)ModifiedLowryProteinAssay(HancockLab)TheadvantageofthisassayovertheLowryassayfromwhichitisderived,isthepresenceofSDSinreagentA.Thisallowsonetoperformproteinassaysinnearlyanydetergent(eg.,whendetergentsareusedinthepurificationofoutermembraneproteins).Inaddition,SDSseparatesmembraneproteinsfromcontaminatingmembraneconstituentsanddenaturestheproteinsallowingmorereproducibleresults.Micro-ProteinAssay(JimBrownLab)Thismicro-proteinassayisnotasaccurateasothermethodsforassayingproteinconcentration,butithasthedistinctadvantageofrequiringonlytraceamounts(lessthan1ug)ofyourvaluableprotein.Inmanycasestheaccuracy(withinafactorof2)oftheassayismorethansufficient.AnotheradvantageoftheassayisitsrelativeeaseandquicknessPhosphoaminoAcidAnalysisofnon-TCAPrecipitableProteins(WoodgettLab)ProteinConcentrationofLaemmliGelSamples(WoodgettLab)
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