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CY-1081

1. Cat CY 1080 CycLex Pyk2 Kinase Assay Inhibitor Screening Kit Cat CY 1081 CycLex FGFR2 Kinase Assay Inhibitor Screening Kit Cat CY 1082 CycLex c Src Kinase Assay Inhibitor Screening Kit Cat CY 1083 CycLex p56Lck Kinase Assay Inhibitor Screening Kit Cat CY 1084 CycLex TrkA Kinase Assay Inhibitor Screening Kit Cat CY 1091 CycLex EphA2 Kinase Assay Inhibitor Screening Kit Cat CY 1092 Tyrosine Kinase Positive Control Weel Positive Control Cat CY E1172 Met Positive Control Cat CY E1080 Pyk2 Positive Control Cat CY E1081 FGFR2 Positive Control Cat CY E1082 c Src Positive Control Cat CY E1083 p56Lck Positive Control Cat CY E1084 TrkA Positive Control Cat CY E1091 EphA2 Positive Control Cat CY E1092 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cydlex c jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 1081 14 Version 140318
2. s where samples or reagents are e Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations e Avoid contact with Substrate Solution which contain e Avoid contact with Stop Solution which Oi Acid e In case of contact with the Stop Solutio ubstrate Solution wash skin thoroughly with water and seek medical attention when e Biological samples may be vii with infectious agents Do not ingest expose to open r r drogen peroxide wounds or breathe aerosols W tive gloves and dispose of biological samples properly e CAUTION Sulfuric Acid ng acid Wear disposable gloves and eye protection when handling St ti Q C CY 1081 5 Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Due to technical reason this kit is adjusted to measure kinase activity of the recombinant catalytic domain of Pyk2 CycLex Pyk2 Positive Control not provided CY E1081 should be used in all assay Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparatio
3. which is efficiently phosphorylated by Pyk2 in vitro Applications of this kit include 1 Screening inhibitors or activators of recombinant catalytic domain of Pyk2 2 Detecting the effects offphatmacological agents on recombinant catalytic domain of Pyk2 This assay kit is for researchjuse only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt Store all components at 4 C e Don t expos ifeagents to excessive light Cat CY 1081 1 Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Intracellular signal transduction following extracellular ligation by a wide variety of surface molecules involves the activation and tyrosine phosphorylation of protein tyrosine kinases PIKs Tyrosine phosphorylation controlled by the coordinated actions of protein tyrosine phosphatases and tyrosine kinases is a critical regulatory mechanism for various physiological processes sneluding cell growth differentiation metabolism cell cycle regulation and cytoskeleton function The focal adhesion PTK family consists of the focal adhesion kinase FAK and the Pyk2 kinase also known as RAFTK CAK beta and CADTK Pyk2 kinase can be activated in response to various stimuli such as TNF alpha changes in osmolarity elevation in intracellular calcium concentration lysophosphatidic acid in addition of brady
4. with disposable tips Precision repeating pipettor e Wash bottle or multichannel dispenser for plate washing e Microcentrifuge and tubes for sample preparation e Vortex mixer e Plate reader capable of measuring absorbance in 96 wells plate at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelengthiof 450 nm which will give a somewhat higher reading e Reagent reseryoirs Deionized water of the highest quality Cat CY 1081 4 Version 140318 4 Pyk2 Kinase Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Store the ATP at 20 C in aliquots Store all other components at 4 C Do not expose reagen excessive light Avoid freeze thaw cycles e Allow all the components to come to room temperature before use e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware Qy e Use deionized water of the highest quality G e Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or ot s Care should be taken to avoid direct contact with these reagents e Do not mouth pipet or ingest any of the reagents e Do not smoke eat or drink when performing the assay handled
5. 6 1997 4 Lev S Moreno H Martinez R Canoll P Peles E Musacchio J M Plowman G D Rudy B Schlessinger J Protein tyrosine kinase PYK2 involved in Ca 2 induc d regulation of ion channel and MAP kinase functions Nature 376 737 745 1995 5 Dikic I Tokiwa G Lev S Courtneidge SA Schlessinger J A role for Pyk2 and Src in linking G protein coupled receptors with MAP kinase activation Nature 383 547 550 1996 6 Takaoka A Tanaka N Mitani Y Miyazaki T Fujii H Sato M ovarik P Decker T Schlessinger J Taniguchi T Protein tyrosine kinase Pyk2 mediates the Jakedependent activation of MAPK and Statl in IFN but not IFN x signaling EMBO J 18 2480 2488 1999 7 Pandey P Avraham S Kumar S Nakazawa A Place A Ghanem L Rana A Kumar V Majumder PK Avraham H Davis RJ Kharbanda S Activation ofp38 mitogen activated protein kinase by PYK2 related adhesion focal tyrosine kinase dependentmechanism J Biol Chem 274 10140 10144 1999 8 Tokiwa G Dikic I Lev S Schlessinger J Activation of Pyk2 by stress signals and coupling with JNK signaling pathway Science 273 792 794h996 Cat CY 1081 13 Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit F 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products Tyrosine Kinase Assay Kit CycLex Weel Kinase Assay Inhibitor Screening Kit Cat CY 1172 CycLex Met Kinase Assay Inhibitor Screening Kit
6. cause an inhibitory effect on Pyk2 actitity the level of A450 is weakened as compared with Solvent control The high level of A450 is not observedin Inhibitor control usually A450 lt 0 5 Assay reagents Test sample poeta ee Kinase Reaction Buffer 80 pL 80 pL 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 pL 10X Staurosporine 100 uM 10 pL CycLex Pyk2 Positive Control 0 1 unit uL 10 uL 10 pL 10 pL 10X Staurosporine 100 uM See page 4 section Materials Required but not Provided Pyk Positive Control 0 1 unit uL See page4 section Materials Required Provided 1 Following the above table add the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of Diluted yeLex Pyk2 Positive Control to each well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Cat CY 1081 Version Pyk2 Kinase Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results 1 Average the absorbance values for the Pyk2 sample duplicates Positive Control and all experimental sample duplicate values when applicable When the Pyk2 Positive Control 1 unit assay is included as an internal control for the phosphorylation reaction the abso
7. cetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit is designed to determine non isotopic kinetic analysis of the Pyk2 catalytic domain kinase activity Careful attention to operation and the assay protocol will provide the investigator With reliable tool for the evaluation of inhibitor or activator of Pyk2 kinase Summary of Procedure Add 100 uL of reaction mixtureito the wells 4 In tibate for 60 min at 30 C Wash the wells 4 Add 100 uL of HRB conjugated anti phosphotyrosine antibody y Incubate for 60 min at room temp Wash the wells t Add 100 uL of Substrate Reagent Add 100 uL of Stop Solution t Measure absorbance at 450 nm Cat CY 1081 3 Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate The following components are suppliedgand are sufficient for the one 96 wells microtiter plate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in afforl zip lock bag with a desiccant pack Wells are coated with recombinant Tyrosine kinase substrate 1 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Kinase Buffer One bottle containing 20 mL of 1X buffer used f
8. ion 7 Pipette 100 AE HRP conjugated Detection Antibody into each well cover with a plate sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate 8 Wash wells five times with Wash Buffer making sure each well is filled completely Remove Cat CY 1081 T Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures yclex residual Wash Buffer by gentle tapping or aspiration 9 Add 100 pL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 5 15 minutes 10 add 100 uL of Stop Solution to each well in the same order as the previously add d Substrate Reagent 11 Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wavelength can be used Wells must be read within 30 mintites of adding the Stop Solution Recommendations Special considerations when screening activators and inhibitors In order to estimate the inhibitory effect on Pyk2 activity in the test chemicals Correctly it is necessary to conduct the control experiment of Solvent control at least once fofjeyery experiment and Inhibitor control at least once for the first experiment in addition to Test sample as indicated in the following table When test chemicals
9. kinin a neuropeptide hormone that binds to a G protein coupled receptor and in turn stimulates phosphatidylinositol hydrolysis Pyk2 kimase is expressed mainly in the central nervous system and in cells derived from hematopoietic lineages 123 while FAK is widely expressed in various tissues and links transmembrane integrin feceptdrs to intracellular pathways Lev et al showed that the Pyk2 protein undergoes rapid tyrosine phosphorylation in response to various stimuli Pyk2 is also tyrosine phosphorylated following activation of the nicotinic acetylcholine receptor by membrane depolarization and by treatment of cells witha caleium ionophore 4 It has been proposed that Pyk2 may represent an important signaling intermediate between neuropeptide activated receptors or neurotransmitters that increase calcium flux jand the downstream signals that regulate neuronal activity Pyk2 may also provide a mechanism for a Variety of short and long term calcium dependent signaling events in the nervous system 2 4 Activation of Pyk2 leads to the modulation of ion channel funhctiom and activation of the MAP kinase signaling pathway including p38 cacade 5 7 Furthermor overexpression of Pyk2 led to activation of JNK and a dominant negative mutant of Pyk2 interfered with ultraviolet light or osmotic shock induced activation of JNK Pyk2 represents a cell type specific stress sensitive mediator of the JNK signaling pathway 8 Measurement of Pyk2 Kina
10. ly after wash Reagent Stability All of the reagents included in the CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit have been testedwfor stability Reagents should not be used beyond the stated expiration date Upon receipt kit redgents should be stored at 4 C Coated assay plates should be stored in the original foil bag sealed by thejzip lock and containing a desiccant pack For research us only not for use in diagnostic or therapeutic procedures Cat CY 1081 9 Version 140318 my Pyk2 Kinase Assay Inhibitor Screening Kit GA ycl ex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant Pyk2 catalytic domain enzyme reaction 2 00 A450 0 0 1 0 2 0 3 0 4 0 Pyk2 dose unit V Fig 2 Time course of recombinant Pyk2 catalytic d nzyme reaction 2 0 A450 0 20 40 60 80 100 Reaction time min C CY 1081 10 Version 140318 wn Pyk2 Kinase Assay Inhibitor Screening Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 1 Dose dependency of ATP recombinant Pyk2 catalytic domain Dose dependency of ATP 2 00 A450 0 20 40 60 ATP conc uM gt 100 Fig 3 2 Km for ATP recombinant Pyk2 catalytic dom 5000 0
11. n of Working Solution 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer provided to 900 mL of ddH O Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH O to the viakof 20X ATP provided lyophilized Mix gently until dissolved The final concentration of the 20X ATP Solution should be 1 25 mM Store the solution in small aliquots e g 100 uL at 20 C 3 Prepare Kinase Reaction Buffer by mixing following reagents 96 assays 10 assays 1 assay Kinase Buffer provided 9 5 mL 950 PL 95 pL 20X ATP Solution 0 5 mL 50 pL 5 uL Total LOmL 1000 pL 100 pL You will need 80 90 uL of Kinase Reaction Bufferper assay well Mix well Discard any unused Kinase Reaction Buffer after use Standard Assay 1 Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells toithe foil pouch refold seal with tape and store at 4 C 2 Prepare the Kinase Assay Buffer containing test chemicals and tyrosine kinase inhibitor see page 8 All assays should be done n d plic te W Add 10 uL of 0 1 unit uL Pyk2 Positive Control or serial dilution of Pyk2 Positive Control to the wells of the assay plate on ice 4 Begin the kifiase reaction by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer and incubate at 30 C fo
12. o not exceed 0 25 units for the blank no enzyme control or 2 5 units for the Pyk2 Positive Control Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of the Pyk2 Positive Control perform a second reading at 405 nm A new O D values measured at 405 nm is used to determine Pyk2 activity ofgoff seale samples The readings at 405 nm should not replace the on scale readings at 450 nm Kinetic Assay 1 Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foulpouch refold seal with tape and store at 4 C N Prepare all samples diluted with Kinase Buffer as needed All samples should be assayed in duplicate w Add 10 uL of 0 1 unit uL Pyk2 P sitive Control or serial dilution of Pyk2 Positive Control to the wells of the assay plate on ice 4 Begin kinase reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervals suggested interval is 5 minutes but should be individually determined for each system After the final addition incubate at 30 C for 20 minutes 5 Stop the reaction by flicking out the contents Alternatively the reaction may be terminated by the addition of 150 uL 0 1 M Na EDTA pH 8 0 to each well 6 Wash wells fivestimes with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspirat
13. or Kinase Reaction Buffer and sample dilution 20X ATP One vial of lyophilized ATP Nay salt HRP conjugated Detection Antibody One bottle containing 12 mL of MRP horseradish peroxidase conjugated anti phosphotyrosine monoclonal antibody PY 39 Ready to use Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2SO Ready to use Materials Required but not Provided Pyk2 Positive Control One vial contains 100 units recombinant catalytic domain of Pyk2 The Positive Control should be added to the first well at 1 unit well For instance in the case of 100 units 100 uL Pyk2 Positive Control diluted Positive Control 1 10 with Kinase Buffer use 10 uL for 1 assay The Positive Control is separately delivered with a dry ice from other kit components The Positive Control should be stored in aliquots at below 70 C Avoid repeated freeze and thaw e 10X Staurosporine 100 uM Staurosporin is available from Sigma Cat S 4400 10 mM stock solution DMSO diluted 1 100 in Kinase Buffer 10X 2 5 MeC methyl 2 5 dihydroxycinnamate 100 uM 2 5 MeC Kyowa Medix Japan Cat OP11 make 5 mM DMSO solutionsgand diluted 1 50 in water e 10X Quercetin dihydrate 500 uM Quercetin dihydrate Sigma Cat Q 0125 make 10 mM DMSO solution and diluted 1 20 in water e Pipettors 2 20 uL 20 200 wand 200 1000 uL precision pipettors
14. p Pyk2 Kinase Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring Pyk2 Kinase Activity CycLex Pyk2 Kinase Assay Inhibitor Screening Kit Cat CY 1081 Intended Use cccccccccccccccceceeeseeeeseees 1 SION n Gs E E 1 Tntroduction ccc cccccccesecssssesesseeesneees 2 Principle of the Assay 3 Materials Provided ccccceeceeeeeseeeeeees 4 Materials Required but not Provided 4 Precautions and Recommendation 5 Detailed Protocol cccccececceseseeeeeeeees 6 8 Evaluation of Results cccccceeeeeeeeeeee 9 Assay Characteristics 9 TroubleshO OU gs isiciacsssiussnccatednnieteaasssusssarnees 9 Reagent StADUI Cy ccccacieisessasasepceicceaneraasconnies 9 Example of Test Result8cccccsssccssassvsnseccccsens 10412 References vesscseici cisisistadsasveceesdesvaceosasanssdevenes 13 Related Products cccccccccceeesseeseeeeees 14 Intended Use The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit is designed to measure the activities of recombinant catal fic domain of Pyk2 Proline rich Tyrosine Kinase 2 for the rapid and sensitive evaluation of inhibitors r activators The phosphotyrosine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in recombinant Tyrosine kinase substrate bt
15. r 60 minutes 5 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 6 Pipette 100 uL HRP conjugated Detection Antibody into each well cover with a plate sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate Cat CY 1081 6 Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 7 Wash wells five times with Wash Buffer making sure each well is filled completely Remoye residual Wash Buffer by gentle tapping or aspiration 8 Add 100 uL of Substrate Reagent to each well and at room temperature ca 25 C for 5 15 minutes 9 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 10 Measure absorbance in each well using a spectrophotoPyk2ric plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Readithe plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes f adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performarice After the last wash remove any remaining Wash Buffer by aspirating or decanting Imyentiithe plate and blot it against clean paper towels Note 2 Reliable signals are obtained when either O D values d
16. rbance value should be greater than l 0 with a background less than 0 2 2 For kinetic analysis on graph paper plot the mean absorbance values for each of the time points on the Y axis versus the time of each reaction minutes on the X axis Assay Characteristics The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit has been shown to detect the kinase activity of recombinant catalytic domain of Pyk2 The assayyshows good linearity of sample response Troubleshooting 1 The Pyk2 Positive Control should be run in duplicate using th protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 The reaction curve is nearly a straight line if the kinetics of the assay is of the first order Variations in the protocol can lead to non linearity of the curve asean assay Kinetics that are other than first order For a non linear curve point to point or quadratic curve fit methods should be used 3 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 4 Overall low signal may indicate that desiccationgof the plate has occurred between the final wash and addition of Substrate Reagent Do not allow she plate to dry out Add Substrate Reagent immediate
17. se activity The protocol generally regarded as most sensitive for the quantitative measurement of Pyk2 kinase activity involves incubation of the Pyk2 Rinase sample with substrate either a natural or synthetic polypeptide such as poly Glu Tyr 4 1 in the presence of Mg Mn and P labeled ATP The reaction is terminated by spotting a sample onto a filter paper disc followed by immersion in acid to precipitate the radiolabeled product The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted While sensitive this method is labor intensive generates hazardous radioactive waste and depends on a radioisotope of short half life It is particularly unsuitable when Kinase assays are only performed on an infrequent basis The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit uses a horseradish peroxidase coupled anti phosphotyrosine monoclonal antibody as a reporter molecule in a 96 wells ELISA format This assay provides a non isotopic sensitive and specific method to detect kinase activity of recombinant Pyk2 catalytie domain Cat CY 1081 2 Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit is a single site semi quantitative immunoassay for kinase acti
18. vity of recombinant catalytic domain of Met Plates are pre coated with a newly designed Tyrosine kinase substrate 1 which can be easily phosphorylatedyby recombinant catalytic domain of Met The detector antibody is PY 39 an antibody that specifically detects the phosphotyrosine residue on Tyrosine kinase substrate 1 The CycLex Research Product CycLex Pyk2 Kinase Assay Inhibitor Screening Kit might be used to follow the kinetics of recombinant catalytic domain of Pyk2 as well as screening Pyk2 imbhibitor or activator To perform the test the recombinant catalytic domain of Pyk2 is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate Tyrosine kinase substrate 1 on the wells in the presence of Mg Mn and ATP The amount of phosphorylated Tyrosine kimase substrate 1 is measured by binding it with a horseradish peroxidase conjugate of PY 39 say antisphosphotyrosine monoclonal antibody which then catalyzes the conversion of the chr mogenic substrate tetra methylbenzidine TMB from a colorless solution to a blue solution or yelow after the addition of stopping reagent The color is quantified by spectrophotometry and reflect the wrelative amount of Pyk2 kinase activity in the sample For kinetic analysis the recombinant catalytic domain of Pyk2 is added to the wells in a similar fashion and at varying times the reaction is stopped bathe addition of a chelator sodium ethylenediaminetetraa
19. y 45 409x 134 72 R 0 9984 4000 0 3000 0 AN gt S o V 2000 0 Km for ATP 2 96uM 1000 0 Version 140318 LA Pyk2 Kinase Assay Inhibitor Screening Kit YCLEX User s Manual A For Research Use Only Not for use in diagnostic procedures Fig 4 Effect of broad spectrum kinase inhibitor Staurosporine on activity of recombinant Pyk2 catalyti domain enzyme reaction 125 0 100 0 Y 50 0 Relative Intensity 25 0 0 0 0 0001 0 001 0 01 0 1 1 10 100 Staurosporine conc uM Version 140318 Pyk2 Kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References j Menegon A Burgaya F Baudot P Dunlap DD Girault JA Valtorta F FAK and PYK2 CAKbeta two related tyrosine kinases highly expressed in the central nervous system similarities and differences in the expression pattern Eur J Neurosci 11 3777 3788 1999 2 Girault JA Costa A Derkinderen P Studler JM Toutant M FAK and PYK2 CAKbeta in the nervous system a link between neuronal activity plasticity and survival Trends Neuros en22 257 263 1999 3 Gismondi A Bisogno L Mainiero F Palmieri G Piccoli M Frati L Santoni A Proline rich tyrosine kinase 2 activation by beta integrin fibronectin receptor cross linking and association with paxillin in human natural killer cells J Immunol 159 4729 473

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