PKB(AKT)KinaseAssayKits,TypeII
KITCOMPONENTS
A.Rabbitanti-phosphosubstrate(anti-pSubstrate):250μg/mLin200μLofTrisacetatebuffer,affinitypurifiedrabbitpolyclonal,anti-phosphopeptidesubstrate(RPRAApTF-NH2)antibodies(Catalog#ICP0190).Theanti-pSubstrateantibodiesrecognizespecificallythephosphorylatedformthesubstrate(RPRAApTF-NH2)ony,butnotthenon-phosphorylatedsubstrate(RPRAATF-NH2).Dilutetheantibodyto0.25-0.5μg/mLwithantibodydilutionbuffer(B)asworkingsolution.Enoughforpreparationof100-200mLofworkingantibodysolution.
B.5xAntibodyDilutionBuffer(AbDB):2x10mL.Dilute10mLofAbDBto50mLwithdistilledwaterforworkingsolution.
C.BSA-SubstrateConjugates(BSA-Sub):Catalog#ICP0316.250μg/mLin600μLkinaseassaydilutionbuffer(D).Approximately10moleculesofkinasesubstratepeptide(RPRAATF-NH2)wereconjugatedtoonemoleculesofBSA.Apparentmolecularweightofthemajorconjugateis70-80KD.TheapparentMWfortheaggregatedformisapproximately150kd.Enoughfor100assays.
D.KinaseAssayDilutionBuffer(ADB):10mL.MOPS,20mM,pH7;beta-Glycerolphosphate,25mM;EGTA1mM;sodiumorthovanadate1mM;DTT1mM;MgCL2,7mM.
E.ATP:2mg,Dissolvein2mLkinaseassaydilutionbuffer(D)forworkingsolution.Maynotbestableinsolution.
Additionalreagentsrequiredbutnotsupplied.
1.Anti-rabbitIgssecondaryantibodies.
2.PBStwashingbuffer
3.3%BSAor3%skimmedmilkasblockingbuffer
4.AllreagentsandmaterialsrequiredforSDS-PAGE,transferandsignalgeneratingsystem(suchasBCIP/NBTorECL).
KITSDESCRIPTION
Qualitycontrol:ThekitsweretestedusingPKBalpha,betaandgama.Sensitivityisapproximately10ngofactiveGST-PKBgama/assay.
StorageandStability:Stablefor6monthsat4℃fromdateofshipment.Avoidthelightandheat.
Descriptionsoftheassay:ThePKB(AKT)kinaseassaykit(TypeII)isanon-radioactive,homogenous,simple,immunoblottypeofassay.ThistypeofassayisdesignedfortheassayofPKBpurifiedonsolidaffinitymatricessuchasIPkinasesorGST-PKBsonGSHbeads.ItisalsousefulfortheactivityanalysisofPKBinthesolutionphase.Theprincipleoftheassayissimple.PurifiedPKBonthesolidmatrixwillphosphorylatethekinasesubstrate-BSAconjugates(componentC)inthesolution,inthepresenceofATP.ThephosphorylatedBSA-substrateisthenanalyzedusingSDS-PAGEresolutionandimmunoblotingtechnique.
SummaryoftheAssay:
1)Kinasereaction:BSA-RPRAATF-NH2+ATP+Kinase→ BSARPRAApTF-NH2
2)SDS-PAGEresolutionandtransfer
3)SpecificimmunoblottingfordetectionofthekinaseproductBSA-RPRAApTF-NH2probedwithanti-PRAApTF-NH2(anti-pSubstrate)
QUALITYCONTROLTEST
AssayforactivityofGST-PKBgamausingthetypeIIkit(ICP0243)
A,125ng;B,60ng;C,30ng;D,15ng;E,7.5ng;F,0.00ng
PROTOCOL
StageOne:Preparationofkinase
1.PreparationofKinaseinSolution:Preparethepurifiedorpartiallypurified(fractionated)PKBin10μLofthekinaseassaydilutionbuffer(componentD)inamicrocentrifugetubes.Forinhibitorscreening,serialdilution(rangefrom500to25ng/assay)ofkinaseshouldbetestedfortheoptimalworkingconcentration.
2.PreparationoftheImmunoprecipitatedKinase:Incubateanti-PKB(abletoIPnativekinase),oranti-GST,anti-HA(fortag-kinase)with10μLto20μLofproteinAforapproximately1hr.Thenwashawayanyunboundantibodies.Incubatethecelllysatein250μLoflysatebufferwitharotorshaker,at4℃for2hrs.Customersshouldselecttheirowncelllysatebufferthatshouldcontainproteaseandphosphataseinhibitors.CentrifugeandaspiratewithPBSttwicethenwithkinaseassaydilutionbuffer(D)twice.Afteraspiration,re-constitutetheimmunoprecipitatedmatrixin10μLofkinaseassaydilutionbuffer(D).
3.PreparationofKinasePurifiedwithAffinityMatrix(GSH,Nicole,maltoseagarose):ThecustomershouldpurifytherecombinantkinasessuchasGST-PKBfollowingtheirownprotocol.Recommend10-20μLofaffinitymatrix/assay.Finalwashandaspirationofthekinaseonmatrixwithkinaseassaydilutionbuffer(D)Thenre-constitutetheaspiredmatrixwith10μLofthekinaseassaydilutionbuffer.
StageTwo:PKBkinaseactivityassay
1.Add5μLoftheBSA-kinasesubstrate(componentC)tokinasepreparationinstageone.
2.Add5μLoftheATP(componentE)solutiontoinitiatethekinasereactionat30-35oCfor60minswithconstantshakingorshakeevery5-mins.
3.Stopthekinasereactionwith20μLofSDS-sampleloadingbufferandboilfor2mins.
StageThree:RunningSDS-PAGEandtransfer
1.Preparea8%SDS-acryamidegel.
2.Add10uL/wellofthereactionsample(fromstagetwo,step3).RuntheSDS-PAGEat100mVfor60min,approximately2cminrunningdistancefromthetopoftheseparatinggeltodye-front.
3.Performthegeltransfertonitrocellulosemembrane(coverthetopofseparatedgelandMWMarker70kd)at200mMfor25mins.
StageFour:Immunoblotting.
1.Blockthemembranewith3%skimmedmilkfor20mins
2.Washthemembranetoremovetheskimmedmilk.
3.Incubatewith20mLofanti-pSubstrate(componentA)workingsolution,atroomtemperaturefor4hours.
4.WashthemembranewithPBSt4timesat5minsinterval.
5.Incubatewithgoatanti-rabbitIgGsecondaryantibodiesfor1-2hours.
4.Customershouldselecttheirownmethods(eithercolormetricorECLsystem)forthesignalgeneration
温馨提示：不可用于临床治疗。
