The EpiQuik™InVivoProteinSumoylationAssayUltraKit isacompletesetofoptimizedreagentsdesignedformeasuringinvivoproteinSUMOylationfrommultiplemammaliancells/tissuesincludinghuman,mouse,andrat. Thekithasthefollowingadvantages: - Fastprocedure,whichcanbefinishedwithin5hours.
- DirectcolorimetricassaywithouttheneedforaffinitychromatographyorWesternblot.
- FlexIBLechoiceofantibodyofinterestallowsforthedetectionofSUMOylationofmultipletargetproteinssimultaneously.
- Useofoptimizeddetectionantibodyeliminatesthestepfordetectionsolutionpreparation,therebyincreasingdetectionsensitivityandassayconvenience.
- Thepositivecontrol(SUMOprotein)andnegativecontrol(un-SUMOylatednon-immuneIgGprotein)allowsproteinSUMOylationtobequantified.
- Stripmicroplateformatmakestheassayflexible:manualorhighthroughputanalysis.
- Reliableandconsistentassayconditions.
BackgroundInformation
SUMOylationisapost-translationalmodificationinvolvedinvariouscellularprocesses,suchasnuclear-cytosolictransport,transcriptionalregulation,apoptosis,proteinstABIlity,responsetostress,andprogressionthroughthecellcycle.SUMOproteinsaresimilartoubiquitin.Thereare3confirmedSUMOisoformsinhumans:SUMO-1,SUMO-2,andSUMO-3.SUMO-2/3showahighdegreeofsimilaritytoeachotherandaredistinctfromSUMO-1.SUMOylationisdirectedbyanenzymaticcascadeanalogoustothatinvolvedinubiquitination.SUMOylationoftargetproteinsinvivohasbeenshowntocauseanumberofdifferentoutcomes,includingalteredlocalizationandbindingpartners.DetectionofinvivoproteinSUMOylation(SUMOconjugation)wouldprovideusefulinformationforunderstandingSUMOmodificationthatemergesasanimportantcontrolmechanismregulatingtheactivityofmanynuclearproteins. Principle&Procedure
Inanassaywiththiskit,theantibodiesspecifictothetargetedproteinsarestablyboundtothestripwellsandthetargetedproteinsarecapturedbytheseantibodies.SUMOylationofthetargetedproteinsaredetectedbyrecognitionofSUMOconjugatedtotheseproteinswithananti-SUMOantibody.TheratioorintensityoftheSUMOylation,whichisproportionaltotheconjugatedSUMOamount,canbequantifiedthroughthesignalreport-colordevelopmentsystembyusingamicroplatereaderat450nmwavelength. StartingMaterials
Inputmaterialisnuclearextract.Theamountofnuclearextractsforeachassaycanbe1µgto20µgwithanoptimalrangeof5to10µg. | 
 Fig.1.SchematicProcedureforUsingtheEpiQuik™InVivoProteinSumoylationAssayUltraKit(Colorimetric).
Fig.2. IllustratedstandardcurvegeneratedwithSUMOproteincontrol. |
