Pierce/c-Met (Soluble) Human ELISA Kit/96 tests/KHO2031

Description

TheHumanc-Met(Soluble)ELISAresearch-use-onlykitisdesignedtodetectandquantifythelevelofsolublehumanc-Metproteininserum,plasma,andcellculturesupernatantsusing96-wellplatesandamicroplatereader.Theassaywillrecognizebothnaturalandrecombinantsolublehumanc-Met.

Performancecharacteristics
•Sensitivity:<0.5>
•Standardcurverange:0.78–50ng/mL
•Sampletypes:serum,plasma,cellculturesupernatants
•Speciescross-reactivity:human
•Samplevolume:100μL(1:2to1:100)
•Totalassayincubationtime:4hrs

Principleofthemethod
TheHumanc-Met(Soluble)kitisasolidphasesandwichenzyme-linkedimmunosorbentassay(ELISA).Ahighlypurifiedantibodyhasbeencoatedontothewellsofthemicrotiterstripsprovided.Duringthefirstincubation,standardsofknownsolublehumanc-Metcontent,controls,andunknownsamplesarePipettedintothewells.Afterwashing,biotinylatedsecondaryantibodyisadded.Afterwashing,streptavidin-peroxidase(enzyme)isadded.Thisbindstothebiotinylatedantibodytocompletethefour-membersandwich.Afterathirdincubationandwashingtoremovealltheunboundenzyme,asubstratesolutionisadded,whichisacteduponbytheboundenzymetoproducecolor.Theintensityofthiscoloredproductisdirectlyproportionaltotheconcentrationofsolublehumanc-Metpresentintheoriginalspecimen.

Backgroundinformation
c-Met,amemberofthetyrosinekinasesuperfamily,isthereceptorforhepatocytegrowthfactor,alsoknownasscatterfactor(HGF/SF).Cellsexpressingc-Metincludeepithelialcells,endothelialcells,bloodcellsofvarioustypes,andglomerularmesenchymalcells.SourcesofHGF/SFincludemesenchymalcells,mesanglialcells,endothelialcells,macrophages,andtumorcells.

HGF/SFbindingtoc-Metstimulatesreceptordimerizationandthephosphorylationofnumerousresidueswithinthereceptor’scytoplasmicdomain.Phosphorylationoftyrosines1234and1235ofc-Metisrequiredforactivationofthereceptor’styrosinekinaseactivity.c-Metphosphorylationalsogeneratesdockingsitesfornumeroussignalingmoleculesandstimulatesreceptorinternalizationviaclathrin-coatedvesicles.Signalingproteinsthatarephosphorylatedand/orlocalizedinresponsetoc-Metphosphorylationinclude:Grb2,Shc,Cbl,Crk,cortactin,paxillin,GAB1,PI-3K,FAK,Src,Ras,ERK1and2,JNK,PLC-γ,AKT,andSTAT3.HGF/SFstimulationofc-Metexpressingcellsenhancesproliferation,migration,morphogenesis,andproteasesynthesis,characteristicsthatareassociatedwithinvasivecellphenotype.

Solublec-Metisatruncatedformofthec-Metmembranereceptor.Itcanbecleavedbyproteasesandreleasedfromthelipidbilayerinaprocessknownasectodomainshedding.Manytransmembraneproteinsarereleasedthroughthissheddingprocessanditisanormalpartofdevelopmentwhichwhendefectivecancauseanumberofpathologies.Thesolubleformofthec-Metreceptorissmallerthanthemembraneboundreceptor,containstheextracellularregionofthereceptor,andisabletobindtheHGFligand.

Detectlow-expressingproteinsanduselesssample
ELISAkitsdesignedtomeasureintracellularsignalingtargetsaretypically2–10timesmoresensitivethanwesternblotting.Theimprovedsensitivityenablesyoutodetectlow-expressingproteinsthatotherwisemaynotbedistinguishablefrombackground.Inaddition,theamountofsampleneededtoruntheassayislessthanwhatisneededforwesternblots.

EachELISAkitisvalidatedforsensitivity,specificity,precision,andlot-to-lotconsistency.Seeproductinsertformoreinformationonvalidation.

Relatedlinks
LearnmoreaboutELISAkits
Learnmoreaboutotherimmunoassays