HTScan™ Profiling Kit ( Set I)

I 8 Kinases Orders I 877-616-CELL (2355) *Various Storage Conditions 12 Reactions/Kinase [email protected] Support I 877-678-TECH (8324) Box 1: Store at -70°C or -80°C [email protected] Box 2: Store at -20°C Web I www.cellsignal.com new 12/04 #7405

Products Included Product # Kit Quantity Storage: Antibodies are supplied in in 10 mM sodium ATP (10 mM) 9804 1 ml HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and DTT (1000x, 1.25 M) 80 µl 50% glycerol. Do not aliquot the antibodies. Store at –20°C. Peptides are supplied at 6 µM in 0.001% Phospho-Tyrosine Mouse mAb (P-Tyr-100) 9411 30 µl DMSO or a DMSO and carbonate buffer solution (15 HTScan™ Tyrosine Kinase Buffer (4x) 9805 15 ml mM Na2CO3, 35 mM NaHCO3, 0.05% NaN3). Store at 8 Tyrosine Kinases 200 U/each –20°C. are supplied in 50 mM Tris-HCL Prearrayed biotinylated substrates 96-well plate (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol. Store at –80°C. Description: The provides a means of characterizing Unit Definition: 10 Units is defined as the amount of the effect of a compound or panel of compounds on the kinase required to maximally phosphorylate 75 pmol of Keep enzymes on ice during use. included active human receptor tyrosine kinases. biotinylated substrate peptide in 30 minutes at 25°C in Avoid repeated freeze-thaw cycles. a total reaction volume of 50 µl quantified by DELFIA® The tyrosine kinases in the kit are supplied as GST to achieve signal-to-noise ratios over 25. See vial label Companion Products: fusion . Biotinylated substrate peptides validated for lot-specific information. Tyrosine Kinase Substrate Screening Kit #7450 for each kinase are supplied at 6 µM, and a phospho- tyrosine monoclonal antibody for detection of the phos- Source/Purification: Each GST-Kinase fusion Serine/Threonine Kinase Substrate Screening Kit #7400 phorylated form of the substrate peptide is also included in this kit was produced using a baculovirus-expressed in the kit. The kit enables rapid characterization of the cDNA fragment with the target human tyrosine kinase HTScan™ EGFR Kinase Assay Kit #7410

effect of a single compound at multiple concentrations domain amino-terminally fused to a GST-HIS6- HTScan™ EPHB3 Kinase Assay Kit #7716 or a panel of compounds toward the following RTKs: Thrombin cleavage site. Proteins were then purified by PDGFRβ, EGFR, FGFR3, VEGFR-2, FLT3, IGF1R, MET one-step affinity chromatography using glutathione- HTScan™ IGFR Kinase Assay Kit #7746 and EPHB3, each representing an RTK subgroup. agarose. HTScan™ MET Kinase Assay Kit #7440 Background: Tyrosine kinases have been validated as The detection antibody, Phospho-Tyrosine Mouse mAb HTScan™ VEGFR-2 Kinase Assay Kit #7788 targets for therapeutic intervention in many disease (P-Tyr-100) #9411, was derived from mice immunized areas. The codes for nearly one hundred with phospho-tyrosine-containing peptides (KLH-cou- HTScan™ FLT3 Kinase Assay Kit #7743 tyrosine kinases, which can be categorized into receptor pled). tyrosine kinases (RTKs) and cytosolic tyrosine kinases. FLT3 (Tyr589) Biotynilated Peptide #1305 Quality Control: Each substrate peptide was selected RTKs are currently a major focus for therapeutic drug Gastrin Precursor (Tyr87) Biotinylated Peptide #1310 target screening in the pharmaceutical industry. using Tyrosine Kinase Substrates Screening Kit #7450. HTScan™ Profiling Kit (Tyrosine Kinase Set 1) from The quality of the biotinylated peptides was evaluated by PYK2 (Tyr402) Biotinylated Peptide #1315 Cell Signaling Technology (CST) is an antibody-based reversed-phase HPLC and by mass spectrometry. IRS1 (Tyr891) Biotinylated Peptide #1320 kinase activity assay kit. The purity of each RTK was characterized using SDS- PAGE, and assay conditions for phosphorylation of the PTP1B (Tyr66) Biotinylated Peptide #1325 Each kinase is paired with a validated biotinylated sub- ® strate peptide, provided in 96-well format. Optimal included substrates were optimized using DELFIA (for BTK (Tyr223) Biotinylated Peptide #1330 ® assay conditions have been identified for each substrate details, visit www.cellsignal.com/drug2.ASP) (DELFIA peptide using a phosphotyrosine-specific antibody to is a registered trademark of PerkinElmer, Inc.). achieve signal-to noise ratios over 25. FGFR2 FGFR3 EphA3 EphA4 EphA5 FGFR1 KDR CSFR/FMS EphA6 Substrate FLT1 EphB1 KIT EphA7 EphB2 Kinase: 1 2 3 4 5 6 7 8 9 10 11 12 Product #: FGFR4 FLT4 PDGFRα EphB3 FLT3 PDGFRβ EphB4 A PDGFRβ 1305 RET

MER EphA8 B FGFR3 1305 MET RON TIE2 AXL TIE1 EphA2 C FLT3 1310 TYRO3/SKY CCK4/ EphA1 TRKC ROR2 RYK PTK7 EphA10 ROR1 EphB6 TRKB FAK D VEGFR-2 1310 TRKA PYK2 MUSK SYK E MET 1315 DDR2 DDR1 ZAP-70 IRR ABL F IGF1R 1320 INSR ARG IGF-1R BMX/ETK ALK BTK G EGFR 1325 LTK ROS TEC H EPHB3 1330 ITK TXK LMR2 FRK SuRTK106 LMR1 LMR3 CTK CSK BLK FES FER SRM BRK TNK1 ACK HCK Assay plate (96 well format): Peptide substrates for the JAK3 HER3 JAK2 LCN LYN indicated eight RTKs have been arrayed in 96-well format. TYK2 JAK1 HER4 FGR SRC FYN YES Tyrosine Kinases HER2 EGFR This product is for research use only and not intended in humans. © 2005 Cell Signaling Technology, Inc. page 1 of 3

Orders I 877-616-CELL (2355) [email protected] Support I 877-678-TECH (8324) [email protected] Web I www.cellsignal.com #7405

120 PDGFRβ FGFR3 100 FLT3 VEGFR-2 80 MET IGF1R 60 EGFR %Inhibition EPHB3 40

20

0 Staurosporine Iressa SU6668 CT53518 Compound panel (5 µM each compound): DELFIA® data were generated using HTScan™ Profiling Kit (Tyrosine Kinase Set I) to detect the inhibition of various RTKs by a panel of compounds (Staurosporine, Iressa, SU-6668 and CT53518). In a 50 µl reaction, 10 Units of each RTK, 1.5 µM substrate peptide, 20 µM ATP and 5 µM inhibitor were used per reaction well (in duplicate) at 25°C for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Kinase IC50(µM) 100 PDGFRβ PDGFRβ 0.68 80 FGFR3 FGFR3 2.4 FLT3 60 VEGFR-2 FLT3 0.043 40 MET VEGFR-2 0.26

% Inhibition IGF1R 20 MET 250 EGFR

0 EPHB3 IGF1R 123

-20 EGFR 240 -10 -8 -6 -4 -2 EPHB3 407 log [SU6668] M

Dose response (SU-6668): DELFIA® data were generated using HTScan™ Profiling Kit (Tyrosine Kinase Set I) to detect the inhibition profile of SU-6668 toward various RTKs. In a 50 µl reaction, 10 Units of each GST-RTK, 1.5 µM substrate peptide, 20 µM ATP and various concentrations of SU6668 were used per reaction well at 25°C for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.) of 3 2 © 2005 Cell Signaling Technology, Inc. page

© 2005 Cell Signaling Technology, Inc. #7405 Suggested Protocol DELFIA I I I I I I Additional SolutionsandReagents(Notincluded) vial. Note: Lot-specificinformationforeachkinaseisprovidedonthe I Suggested KinaseAssayConditions: Life SciencesAAAND-0005) Peptide substratesaresuppliedinH .Setup2xReactioncocktails(Mock/NoKinaseandPlus 4. 3. Transfer12.5µlofsubstratepeptidestoplatewellscontaining 2. Microcentrifugebrieflyat4 Transferenzymesfrom-80 1. .Add25µl2xkinasereactioncocktail(containing kinase)to25 5. DELFIA DELFIA Combine 12.5µlof6µMpeptidesubstrateandH eo) nuaea 5Cfr3 minutes. below). Incubateat25°Cfor30 compound ofinterest.Add2xkinasereactioncocktail(see #1244-105) Sciences #AD0124) DELFIA Stop Buffer:50mMEDTApH8 Bovine SerumAlbumin(BSA) Wash Buffer:1XPBS,0.05%Tween-20(PBS/T) pH 7.5,20mMMgCl 2.5 ml4XHTScan™TyrosineKinaseBuffer(240mMHEPES Kinase) intubesonice. tom ofthevial.Returnimmediatelytoice. Bring to5mlwithH 10 µlDTT(1.25M) 20 µlATP(10mM) enzyme tothawonice. 12.5 µlofdilutedcompoundsorH µl dilutedpeptideineachwell.Mixbygentleagitation. cocktail. Immediatelyproceedtostep5. concentration shouldbe10Units/25µlin2Xreaction enzyme Transfer eachenzymeto0.5ml2Xreactioncocktail.Final ® is aregisteredtrademarkofPerkinElmerLifeSciences ® ® ® Enhancement Solution(PerkinElmerLifeSciences Europium-labeled Anti-mouseIgG(PerkinElmerLife Streptavidin coated,96-well,yellowplate(PerkinElmer Protocol forHTScan™ProfilingKit(TyrosineKinaseSet1) 2 2 0. Mixgentlybyinversion , 20mMMnCl ° ° C immediatelytoice.Allow C tobringallliquidthebot- 2 O plus0.001%DMSO. 2 0. 2 , 12 µ M Na 3 VO 4 ) 2 O with Final AssayConditionsfora50 8 Detect615nmfluorescenceemissionwith340excitation 18. Incubateatroomtemperaturefor5minutes. 17. 6 Add100µl/wellDELFIA 16. *Washfivetimeswith200µl/wellPBS/T. 15. Mixandincubateatroomtemperaturefor30minutes. 14. DiluteEuropiumlabeledanti-mouseIgG1:500inPBS/Twith 13. *Washthreetimeswith200µl/wellPBS/T. 12. 11. Mixandincubateatroomtemperaturewithrockingfor60 10. Diluteprimaryantibody,Phospho-TyrosineMonoclonal gation ofplateswhenappropriate,greatly improvesreproducibility. *IMPORTANT: Useofanautomatedmicroplatewasheraswellcentrifu- .Transfer25µlofeachreactionand75H 8. Add50µlStopBuffer(50mMEDTA,pH8)toeachwelland 7. Incubatereactionplateat25°Cfor30minutes. 6. .*Washthreetimeswith200µl/wellPBS/T. 9. information. Email:[email protected] formulated andcarrier-free), anddetailedpeptidesubstratesequence Please contactCellSignalingTechnology forHTS-readyantibodies(PBS mix bypipetting. 10 UnitsKinase using appropriateTime-ResolvedPlateReader(400µSdelay). 1.5 20 µMATP 1.25 mMDTT 3 5 mMMnCl 5 mMMgCl 60 mMHEPESpH7.5 1% BSA.Add100µldilutedantibody/well. minutes. Add 100µlprimaryantibodyperwell. Antibody (P-Tyr-100)#9411,1:1000inPBS/Twith1%BSA. 60 minutes. streptavidin-coated plateandincubateatroomtemperaturefor µ M Na µ M peptide 3 VO 2 2 4 ® Enhancement Solution. µ l Reaction 2 O/well toa96-well

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