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AlphaKetoglutarate(alphaKG)AssayKitprovidesasimple,sensitiveandrapidmeansforquantifyingalphaKG.
InthealphaKGassayprotocol,alpha-ketoglutarateistransaminatedwiththegenerationofpyruvate,whichisusedtoconvertanearlycolorlessprobetoacolored(λmax=570nm)andfluorescent(Ex/Em=535/587nm)product.
AlphaKetoglutarateassayprotocolsummary:-addreactionmixtostandardandsamplewells-incubatefor30min-analyzewithamicroplatereader
ThisproductismanufacturedbyBioVision,anAbcamcompanyandwaspreviouslycalledK677Alpha-KetoglutarateColorimetric/FluorometricAssayKit.K677-100isthesamesizeasthe100testsizeofab83431.
Standardcurve:meanofduplicates(+/-SD)withbackgroundreadssubtracted
ab83431被引用在34文献中.
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AskedonMar022012
VielenDankfürIhrenAnruf.DasLaborhataufunsereAnfragehinbesttigt,dasseswichtigist,dieZellenmechanischaufzuschlieen,d.h.zuhomogenisieren.WieSierichtigsagten,reichtPBSdafürnichtaus,sondernesempfiehltsicheineHomogenisierungmiteinemDounceHomogenizer.VielenDank,dassSieunsaufdieseUnklarheithingewiesenhaben.WirwerdendasProtokollinKürzeaktualisieren.Ichhoffe,dieseInformationenhelfenIhnenweiter,undichwünscheIhnenvielErfolgfürIhrProjekt.BittezgernSienicht,sichwiederbeiunszumelden,fallsSieweitereFragenhaben.
回复于Mar022012
AskedonJul102013
Themostefficientresultswillbehadifthehomogenateismadewiththeassaybufferprovidedwiththekit.Rapidlyhomogenize~2x10^6cellswith100μloficecoldα-KGAssayBufferusingadouncehomogenizer.Deproteinatethesamplessoyoucangetridofanyinterferingproteinsincludingtheenzymes.Thisisasmallmoleculeanalyteassayandcellularenzymescaninterferewiththesignaldevelopment
KeithB
回复于Jul102013
AskedonMay292013
Therearetwooptionsforthesamplepreparation.Eitherjustrapidlyhomogenizecellswith100μloficecoldPBSorotherbuffer(pH6.5-8)andthenadd1-50μlsamplesintoduplicatewellsofa96-wellplateandbringvolumeto50μlwithAssayBuffer.Or:Topreventenzymesinterferingwiththeassayusetheperchloricacid/KOHprotocol(stepatoe).Forcells,Icanrecommendtopelletthemandthenfreezetheminliquidnitrogen.
回复于May292013
AskedonFeb142013
Thankyouforcontactingus.DidyoucomparethesignalyouobtainedfromtheoxaloacetatewiththeresultsfromanegativecontrolThecontrolwerecommendistestingwithoutadditionoftheconvertingenzyme,inordertoaccountforsignalcontributedbypyruvateandotherprobereactants,andsubtractingthisvaluefromthevaluesobtainedfromthesamplesthatareincubatedwiththeconvertingenzyme.
回复于Feb142013
AskedonJun192012
Thankyouforyourinquiry.Yes,bufferswithpHrange6.5–8canbeused.JustmakesurethattheRIPAbufferiswithintherecommendedpH.Ihopethisinformationhelps.Pleasecontactuswithanyotherquestions.
回复于Jun192012
AskedonJun052012
回复于Jun052012
AskedonOct262011
Thisisafluorescentassayandyouwillhavetomeasureattwowavelengths:535nmand587nm.Ifyouonlyhavethe595nmandnotthe535nmyouwillnotbeabletousethefluorescentmeasurements.However,thisassaycanalsobemeasuredcolorimetrically(lmax=570nm).Soifyour595nmfilterhasabandwidthoptionof+/-25,youcanmeasureattherequired570nmandbeabletocapture100%ofthesignal.
回复于Oct262011
AskedonJul292011
Thiskitcanbeusedtomeasurealphaketoglutarateinblood,plasmaandIsletsofLangerhansincludingtissueculturesupernatants,urine,andserumsamples.
回复于Jul292011
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