For ResearchUseOnly. NotForUseInDiagnostic Procedures. © 2012 Cell Signaling Technology, Inc. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Dg Species Cross-Reactivity Key: Applications Key: GTP hydrolysisisaided byGTPaseactivatingproteins the protein’s intrinsic GTPaseactivity, renderingitinactive. of Rac/Cdc42,andthehydrolysisGTPto GDPthrough and development(5).GTPbindingstimulates theactivity membrane trafficking,transcriptionalregulation, cellgrowth, play keysignalingrolesincytoskeletalreorganization, brain, isalsofoundinmanyothertissues. RacandCdc42 hematopoietic origin,andRac3,whilehighlyexpressedin ubiquitously expressed.Rac2isexpressedincellsof and Cdc42,themostwidelystudiedofthisgroup,are and Rac3,whicharehighlysimilarinsequence.Rac1 In mammals,Racexistsasthreeisoforms,Rac1,Rac2, Rac andCdc42aremembersoftheRho-GTPasefamily. return theGTPasetoitsGDP-boundinactiveform(4). bound formandGTPaseactivatingproteins(GAPs)that factors (GEFs)thatpromoteformationoftheactiveGTP- proteins arealsoregulatedbyguaninenucleotideexchange exchange andGTPhydrolysisactivitiesofRassuperfamily downstream effectors.Theseintrinsicguaninenucleotide activity, whichhydrolyzesGTPtoGDPandreleasesthe activation canbeswitchedoffbytheintrinsicGTPase the bindingandregulationofdownstreameffectors.This to change initsdownstreameffector-binding region,leading activated Gproteinthengoesthroughaconformational of GTPandformationtheGTP-boundform(active).The the GDP-boundform(inactive),whichleadstobinding upstream signalstimulatesthedissociationofGDPfrom and movement,vesicularnucleartransport(1).An actincytoskeletalorganization,cellpolarity cell survival, developmental eventsthatincludecellcycleprogression, switchesindiversecellularand and functionasbinary G proteinshavebothGDP/GTP-bindingandGTPaseactivities similarities: Ras,Rho,Rab,Arf,andRan(1-3).Thesesmall at leastfivefamiliesbasedontheirsequenceandfunctional of proteins(over150members)thatcanbeclassifiedinto GTP-binding proteins(Gproteins)comprisealargeclass Background: also detecthomologousproteinsfromotherspecies. species, asdeterminedthroughin-housetesting,butmay shown inFigure1.Thiskitdetectsproteinsfromtheindicated detects endogenouslevelsofGTP-bound(active)Cdc42as Specificity/Sensitivity: Cdc42 MousemAb. activation levelisthendeterminedbywesternblotusinga then beimmunoprecipitatedwithglutathioneresin.Cdc42 to bindtheactivatedformofGTP-boundCdc42,whichcan GTPase inthecell.GST-PAK1-PBD fusionproteinisused formeasuringactivationofCdc42 the reagentsnecessary Description: Species Cross-Reactivity:H,M #8819 —dog (30 immunoprecipitations) 1 Kit Active Cdc42DetectionKit Pg —pig Active Cdc42DetectionKitprovidesall Background: Sc W
—S. cerevisiae —Western Active Cdc42DetectionKit H The Rassuperfamilyofsmall —human IP Ce —Immunoprecipitation —C. elegans M —mouse Hr —horse R —rat IHC
—Immunohistochemistry Hm The elutedsamplecanthenbeanalyzedbywestern blot. binding protein.Step2:Removeunboundproteins bycentrifugation.Step3:Eluteglutathioneresin-boundGTPasewithSDSbuffer. glutathione resininthespincupandincubate at4ºCtoallowGTP-boundGTPasebindingtheglutathioneresinthroughGST-linked Figure 2.TheGTP-boundGTPasepull-down processcanbedividedinto3stepsasshown.Step1:Mixsample,bindingprotein,and Spin CupandCollectionTubes SDS SampleBuffer Glutathione Resin Lysis/Binding/Wash Buffer Components ShipAs:11860S Cdc42 MousemAb GST-Human PAK1-PBD Components ShipAs:11859S GDP GTP Components ShipAs:11859S —hamster lutat ione inding rotein - ound T ase T - ound T ase t er roteins sample 1 g S 2 All collection tu e Mk —all species expected —monkey 3 spin cup ChIP —Chromatin Immunoprecipitation - incu ate and indingprotein - mi samplewit resin 4 T- K1- T γ Mi rev. 09/24/20 —mink tep 1 Species enclosed inparenthesesarepredicted toreactbasedon100% homology. C —chicken Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.Anti-mouse IgG,HRP-linkedAntibody#7076wasusedasthesecondary µL oftheelutedsamples(lanes2,3,and4)wasperformedusingaCdc42MousemAb. as anegativecontrol(lane4).Western blotanalysisofcelllysate(20µg,lane1)or20 lysate wasalsoincubatedwithoutGST-PAK1-PBD inthepresenceofglutathioneresin incubated withglutathioneresinandGST-PAK1-PBD (lanes2and3).GTP or GDPtoactivateinactivateCdc42(referoptionalstepC).Thelysateswerethen Figure 1.NIH/3T3celllysates(500µLat1mg/ml)weretreatedinvitrowithGTP Dm IF —Immunofluorescence —D. melanogaster LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories. LumiGLO isaregistered trademarkofKirkegaard&Perry X Support —Xenopus Orders Web tep 2 F was —Flow cytometry Item # Item # Item # 11524 11522 11521 11526 11525 11523 8747 8659 Z n n n —zebrafish www.cellsignal.com [email protected] 877-678-TECH (8324) [email protected] 877-616-CELL (2355) Kit Quantity Kit Quantity Kit Quantity 1 X100mL 1 X600µg 1 X30vial 1 X1.5ml 1 X300µl 1 X50µl 1 X50µl 1 X3ml E-P B —ELISA-Peptide —bovine Storage Temp Storage Temp Storage Temp –20°C –20°C –80°C –80°C 4°C 4°C 4°C estern lot RT γ S-treated tep 3 elute γ S
page 1 of 3 © 2012 Cell Signaling Technology, Inc. Background References: Rac andCdc42,intheirinactiveGDP-boundstate(6,7). dissociation inhibitor, whichretainsRhofamilyGTPases,including is achievedthroughthebindingofRhoGDI,aguaninenucleotide nucleotide exchangefactors(GEFs).Anotherlevelofregulation (GAPs), whileexchangeofGDPforGTPisfacilitatedbyguanine (7) (6) (4) (3) (2) (1) (5) Wennerberg, K.andDer, C.J.(2004) Rossman, K.L.etal.(2005) Bernards, A.andSettleman,J.(2004) Vigil, D.etal.(2010) Wennerberg, K.etal.(2005) Colicelli, J.(2004) Takai, Y. etal.(2001) 377-85. Orders n 877-616-CELL (2355)[email protected] Sci STKE Physiol Rev Nat RevCancer Nat RevMolCellBiol 2004,RE13. J CellSci 81,153-208. J CellSci 10,842-57. Trends CellBiol 118,843-6. 117,1301-12. 6,167-80. 14, Support n
877-678-TECH (8324)[email protected] Web n www.cellsignal.com
page 2 of 3 © 2020 Cell Signaling Technology, Inc. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Active Cdc42 Detection Kit_2012 09/20 B A Additional MaterialsRequired D C #8819 NOTE: Add1mMPMSFimmediatelypriortouse. 1X CellLysis/Binding/Wash Buffer 1X PhosphateBufferedSaline(PBS) the followingsteps. NOTE: We recommendmaking1mg/mllysatein1XLysis/Binding/Wash Bufferfor 5. 4. 3. 2. 1. Cell Lysis NOTE: PreparesolutionswithddH Solutions andReagents Tris BufferedSalinewithTween Blue LoadingBufferPack#7722 Phenylmethanesulfonyl fluoride(PMSF)#8553 4. 3. 2. 1. Affinity PrecipitationofActivatedGprotein 4. 3. 2. 1. In vitroGTPγSorGDPTreatment (Optional) 20X LumiGLO Anti-mouse IgG,HRP-linkedAntibody#7076 Biotinylated ProteinLadderDetectionPack#7727 Color-coded PrestainedProteinMarker, BroadRange(10-250kDa)#74124 Milk#9999 Nonfat Dry Bovine SerumAlbumin(BSA)#9998 antibodydilutionbuffer:1XTBST5%BSA Primary 1 MMgCl 0.5 MEDTA, pH8.0#7011 Orders protein assay. for GTPaseassays.Lysate proteinconcentrationcanbedeterminedusingBCA new tube.Thesupernatantisthecelllysate.Freshlysatesarerecommended Microcentrifugeat16,000xg4°Cfor15minandtransferthesupernatanttoa Votex thetubebrieflyandincubateonicefor5min. cells, justresuspendthepellet. Scrape cellsofftheplateandtransfertoanappropriatetube.Fornon-adherent PMSF toeachplate(10cmindiameter),orcellpelletsfrom75 Remove PBSandadd0.5mlice-cold1XLysis/Binding/Wash Bufferplus1mM and resuspendcellsin10mlice-coldPBS. once withice-coldPBS.Fornon-adherentcells,pelletcellsat100xgfor5min To cellsunder nondenaturingconditions,removemediaandrinsecells harvest aliquots forlateruse at -80°C. ThawtheGST-PAK1-PBD oniceandimmediatelymake20µgaliquots. Store 6,000xg for10-30sec.Discardtheflow-through. spin cupwithresin.Invertthetubesgently severaltimes.Centrifugethetubesat Discardtheflow-through.Add400µlof1X Lysis/Binding/Wash Buffertoeach fuge thetubesat6,000xgfor10-30sec. tothespincupwithcollectiontube.Centri Add 100µlofthe50%resinslurry Swirlthebottleofglutathioneresintothoroughlyresuspendagarosebeads. Insert aspincupintocollectiontubeforeachsample. 1 MMgCl Terminate thereactionbyplacingsampleoniceandadding32µlof Incubate themixtureat30°Cfor15minwithconstantagitation. GDP (forafinalconcentrationof1mM),vortexthesample. Add 5µlof10mMGTPγS(forafinalconcentration0.1mM)or100 10 mM),vortexthesample. For 500µllysate,add100.5MEDTA pH8.0(forafinalconcentrationof GDP atfirstusetominimizefreeze/thawcycles. Use 500µgofcelllysateforeachtreatment.Forbestresults,aliquotGTP control), toensuretheimmunoprecipitationproceduresareworkingproperly. Perform thefollowingtreatments,GTP (approx 1-2x10 2 n ® 877-616-CELL (2355)[email protected] Reagentand20XPeroxide#7003 2 (forafinalconcentrationof60mM),vortexthesample. 7 cells). ® 20(TBST-10X) #9997 2
O orequivalentlypurifiedwater. γS (positivecontrol)andGDP(negative Active Cdc42DetectionKitProtocol
Support 2
flask γS and n -
877-678-TECH (8324)[email protected] E 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. membranes, adjustvolumesaccordingly. NOTE: Volumes arefor10cmx(100 Western blotanalysis 11. LumiGLO 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. Incubate membranewith10mlLumiGLO Wash threetimesfor5mineachwith15mlof1XTBST. for 1hratroomtemperature. protein markersin10mlof1XTBSTcontaining5%milkwithgentleagitation and Anti-biotin,HRP-linkedAntibody(1:1000,#7075)todetectbiotinylated Incubate membranewithAnti-mouseIgG,HRP-linkedAntibody(1:2000,#7076) Wash threetimesfor5mineachwith15mlof1XTBST. antibody dilutionbufferwithgentleagitationovernightat4°C. Incubate membranewithCdc42MousemAb(1:167dilution)in10mlprimary Wash threetimesfor5mineachwith15mlof1XTBST. and 5%BSA)for1hratroomtemperature. Incubate membranein25mlofblockingbuffer(TBScontaining0.1%Tween-20 temperature. After transfer, washnitrocellulosemembranewith25mlTBSfor5minatroom Electrotransfer tonitrocellulosemembrane. Tris-glycine gelprovidesbestseparation. or (#7727, 10µl/lane)todeterminemolecularweights.4-20%acrylamide (#74124, 10µl/lane)toverifyelectrotransferandbiotinylatedproteinladder NOTE: CSTrecommendsloadingprestainedmolecularweightmarkers Load 20-25µlontoSDS-PAGE gel(10cmx10cm). signal ismostintenseimmediatelyfollowingLumiGLO proper exposuretime.NOTE:Duetothekineticsofdetectionreaction, wrap andexposetolightfilm.Aninitial10-secexposureshouldindicatethe wrapinplastic Drain membraneofexcessdevelopingsolution(donotletdry), response istoofast. Peroxide, #7003,and9.0mlddH over thefollowing2hr. room temperature.NOTE:LumiGLO phoresed onagelorstoredat-20°Cforfutureuse. Heattheelutedsamplesfor5minat95-100°C.Samplesmaybeelectro containing theresin. Centrifugethetubeat6,000xgfor2min.Removeanddiscardspincup bate atroomtemperaturefor2min. Add50µl2Xreducingsamplebuffertotheresin.Vortex thesampleandincu- 200 mM. adding dithiothreitol(DTT)to2XSDSSampleBufferafinalconcentrationof Prepare50µlofreducingsamplebufferforeachpull-downreactionby Transfer thespincuptoanewcollectiontube. this washsteptwoadditionaltimes. three times,andcentrifugeat6,000xgfor10-30sec.Discardthebuffer. Repeat To washresin,add400µlof1XCellLysis/Binding/Wash Buffer, invertthetube filmandtransferthespincuptoanewcollectiontube. Removethelaboratory Centrifugethespincupwithcollectiontubeat6,000xgfor10-30sec. Incubatethereactionmixtureat4°Cfor1hrwithgentlerocking. may resultfromthepresenceofdetergentinlysate,vortexsample. filmtoprevent leakage,which Sealcapofthecollectiontubewithlaboratory total protein)tothespincup,closecapandvortexsample. Immediatelytransferupto700µlofthecelllysate(containingatleast500µg Add20µgofGST-PAK1-PBD tothespincupcontainingglutathioneresin. ® is a registered trademark of Kirkegaard & Perry Laboratories. isaregisteredtrademarkofKirkegaard& Perry 2 0 water)withgentleagitationfor1minat
® Web substratecanbefurtherdilutedifsignal ® (0.5ml20XLumiGLO 2 n ) ofmembrane;fordifferentsized www.cellsignal.com ® incubationanddeclines
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