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CY-1091

1. Add 100 uL of Stop Solution t Measure absorbance at 450 nm Cat CY 1091 3 Version 140318 TrkA 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 20 mL bottle of 1X buffer used for Kinase Reaction Buffer and Sample dilution 20X ATP One vial of lyophilized ATP Na salt HRP conjugated Detection Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti phosphotyrosine monoclonal antibody PY 39 Substrate Reagent One bottle containing 20 mL of the chromogeniessubstrate 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 TrkA Positive Control One vial contains 100 mits recombinant catalytic domain of TrkA The Positive Control should be added to the first Well at 1 unit well For instance in the case of 100 units 100 uL TrkA Positive Co
2. TrkA dose unit Y Fig 2 Time course of recombinant TrkA catalytic dg zyme reaction 2 5 2 0 A450 0 5 0 20 40 60 80 100 120 140 Time min C CY 1091 10 Version 140318 wn TrkA Kinase Assay Inhibitor Screening Kit Ca yCLeXx User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 1 Dose dependency of ATP recombinant TrkA catalytic domain 2 50 oO 1 50 A450 lt 1 00 0 50 0 00 0 20 40 60 80 100 ATP conc uM LW Fig 3 2 Km for ATP recombinant TrkA catalytic di 4000 0 y 27 439x 256 6 3000 0 gt Rf 0 9993 Km 9 4 uM C CY 1091 11 Version 140318 4 TrkA Kinase Assay Inhibitor Screening Kit ycLex User s Marni For Research Use Only Not for use in diagnostic procedures Fig 4 1 Effect of broad spectrum kinase inhibitor staurosporine on activity of recombinant TrkA catalytic domain enzyme reaction 120 100 80 60 40 Relative Intensity 20 0 001 0 01 0 1 1 10 100 Staurosporine conc nM Fig 4 2 Effect of broad spectrum kinase inhibitor s rine on activity of recombinant TrkA catalytic domain enzyme reaction using radio pe gamma P ATP gt 120 00 100 00 80 00 60 00 40 00 Relative Intensity 20 00 0 00 0 001 0 01 0
3. 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 15 minutes 5 Stop the reaction by flicking out the contents Alternatively the reaction may be terminated by the addition of 450 ub 0 1 M Na EDTA pH 8 0 to each well 6 Wash wells fivedtimes with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 7 Pipette 100 PE HRP conjugated Detection Antibody PY 39 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 1091 7 Version 140318 yelex User s Manual TrkA Kinase Assay Inhibitor Screening Kit For Research Use Only Not for use in diagnostic procedures 8 Wash wells five times with Wash Buffer making sure each well is filled completely Remove 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 added Substrate Reagent 11 Measure absorbance in each well using a spectrophotometric plate reader at dualjwavelengths of 450 540 nm Dual wavel
4. 1 1 10 100 Staurosporine conc nM C CY 1091 12 Version 140318 LA TrkA Kinase Assay Inhibitor Screening Kit YCLEX User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Kaplan D R Hempstead B L Martin Zanca D Chao M V and Parada L F The proto oncogene product A signal transducing receptor for nerve growth factor Science 252 55 1991 2 Klein R Jing S Q Nanduri V O Rourke E and Barbacid M The trk proto oncogei receptor for nerve growth factor Cell 65 189 19 1991 lesa w Barbacid M The trk family of neurotrophin receptors J Neurobiol 25 1386 1403 1994 4 Schneider R Schweiger M A novel modular mosaic of cell adhesion veis Reese domains of the neurogenic trk and trkB tyrosine kinase receptors Oncogene 991 n Kaplan D R and Miller FD Neurotrophin signal transduction in the nervous system Curr Opin Neurobiol 10 381 391 2000 O C CY 1091 13 Version 140318 TrkA 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 Cat CY 1080 CycLex Pyk2 Kinase Assay Inhibitor Screening Kit Cat CY 1081 CycLex FGFR2 Kinase Assay Inhibitor Screening Kit Cat CY 1082 CycLex
5. Research Product CycLex TrkA 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 TrkA CycLex TrkA Positive Control not provided Cat CY E1091 which should be used in all assays Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation 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 texm storage 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH O to the vial of 20X ATP provided lyophilized Mix gently until dissolved The final concentration of the20X 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 uL 20X ATP Solution 0 5 mL 50 uL 5 uL Total 10 mL 1000 pL 100 pL You will need 80 90 uL of Kinase Reaction Buffer per assay well Mix well Discard any unused Kinase Reaction Buffer after use Standard Assay 1 Remove t
6. Substrate Reagent 10 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 Readiithe 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 do not exceed 0 25 units for the blank no enzyme control or 2 5 units for the TrkA Positive Control Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of the TrkA Positive Control perform a second reading at 405 nm A new O D values measured at 405 nm is used to determine TrkA 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 foil potich refold seal with tape and store at 4 C 2 Prepare all samples diluted with Kamas Buffer as needed All samples should be assayed in duplicate w Add 10 uL of 0 1 unit uL TrkA Positive Control or serial dilution of TrkA Positive Control
7. 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 c0 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 1091 14 Version 140318
8. ed by TrkA in vitro Applications of this kit include 1 Screening inhibitors or activators of recombinant catalytic domain of TrkA 2 Detecting the effects of pharmacological agents on recombinant catalytic domain of TrkA 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 1091 1 Version 140318 TrkA Kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction The Trk tropomyosin receptor kinase receptors belong to the family of receptor tyrosine kinases and three trk genes have been identified in mammals The TrkA protooncogene was first identified as an nerve growth factor receptor NGFR 1 2 followed by TrkB and TrkC 3 Nerve growth factor NGF is the preferred ligand for TrkA brain derived neurotrophic factor BDNF and Neufotrophin 4 5 NT 4 5 are preferred for TrkB and Neurotrophin 3 NT 3 for TrkC 3 These specificities ar not absolute and NT 3 is also a ligand for TrkA and TrkB These Trk receptors are transmembrane glycoproteins of 140 kD They are tyrosine kinases with an extracellular ligand binding domain containing multiple repeats of leucine rich motifs two cysteine clusters Cl C2 two immunoglobulin like domains Ig1 Ig2 and a single transmembrane doma
9. engths of 450 550 or 450 595 nm can also be used Read th plate at 450 nm if only a single wavelength can be used Wells must be read within 30minutes of adding the Stop Solution Recommendations Special considerations when screening activators and inhibitors In order to estimate the inhibitory effect on TrkA activity in the test chemicals correctly it is necessary to conduct the control experiment of Solvent control at least onee forevery experiment and Inhibitor control at least once for the first experiment in addition to Test samples as indicated in the following table When test chemicals cause an inhibitory effect on TrkAyactivity the level of A450 is weakened as compared with Solvent control The high level of A450 is notiobserved in Inhibitor control usually A450 lt 0 4 Assay reagents Test sample Solvent control Tahirat control Kinase Reaction Buffer 80 uD 80 uL 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 uL 10X Staurosporine 100 nM 10 uL TrkA Positive Control 0 1 unit ud 10 uL 10 uL 10 pL 10X Staurosporine 100 nM See page 4 Section Materials Required but not Provided TrkA Positive Control 0 1 unit d gt See page 4 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 CycLex TrkA Positi
10. he appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells the foil pouch refold seal with tape and store at 4 C N Prepare the Kinase Assay Buffer containing test chemicals and tyrosine kinase inhibitor see page 8 All assays should be donedn duplicate W Add 10 uL of 0 1 unit tL TrkA Positive Control or serial dilution of TrkA Positive Control to the wells of the assay plate on ice 4 Begin the kifaselreaft n by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer and ieubate at 30 C for 30 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 1091 6 Version 140318 TrkA 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 incubate 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
11. in 4 Mhe tyrosine kinase domains are highly related 80 amino acid identity however the extracellular domains are more divergent 30 Signaling through the Trk receptors the main pathways Neurotrophin bindifig to Trk receptors triggers dimerization leading to the activation of different signaling pathways through recruitment of various adapter molecules The main binding sites for Trk substrates are two tyrosine residues that on activation of the Trk receptors become phosphorylated Two complexes of adapter molecules bind to the tyrosine residue located in the juxtamembrane region of the Trk receptorjythe Shc Grb2 SOS and the FRS2 SHP 2 Grb2 SOS complex Ras Raf MEK MAPK induces the differentiation of neurons and neurite growth 5 Measurement of TrkA Kinase activity The protocol generally regarded as most sensitive for the quantitative measurement of TrkA kinase activity involves incubation of the TrkA kinase sample withysubstrate 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 papezs 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 sho
12. ne 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 ay anti2phosphotyrosine monoclonal antibody which then catalyzes the conversion of the chr6mogenic substrate tetra methylbenzidine TMB from a colorless solution to a blue solution or yellow after the addition of stopping reagent The color is quantified by spectrophotometry and reflect th relative amount of TrkA kinase activity in the sample For kinetic analysis the recombinant catalytic domain of TrkA is added to the wells in a similar fashion and at varying times the reaction is stopped bathe addition of a chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product CycLex TrkA Kinase Assay MInhibitor Screening Kit is designed to determine non isotopic kinetic analysis of the TrkA catalytic domain kinas activity Careful attention to operation and the assay protocol will provide the investigator with areliable tool for the evaluation of inhibitor or activator of TrkA kinase Summary of Procedure Add 100 uL of reaction mixture to the wells 4 Ineubate for 30 min at 30 C Wash the wells t Add 100 uIORHREP conjugated anti phosphotyrosine antibody J Incubate for 60 min at room temp Wash the wells t Ada 100 uL of Substrate Reagent t
13. ntrol 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 aliquotsatbelow 70 C Avoid repeated freeze and thaw 10X Staurosporine 100 nM Staurosporine is available from Sigma Cat S 4400 10 uM stock solution DMSO diluted 1 100 in Kinase Buffer 10X Quercetin dihydrate 500 uM Quercetin dihydrate Sigma Cat Q 0125 make 10 mM DMSO solution and diluted 1 20 in water Pipettors 2 20 uL 20 200 uL and200 1000 uL precision pipettors with disposable tips e Precision repeating pipettor e Wash bottle or multichannel dispenserfor plate washing e Microcentrifuge and tubes for sample preparation e Vortex mixer e Plate reader capable of measunmg absorbance in 96 well plates 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 wavelength of450 nm which will give a somewhat higher reading e Reagent reservoirs Deionized watenrof the highest quality Cat CY 1091 4 Version 140318 4 TrkA 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
14. p TrkA Kinase Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring TrkA Kinase Activity CycLex TrkA Kinase Assay Inhibitor Screening Kit Cat CY 1091 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 ccccccceesseeseeeeees 14 Intended Use The CycLex Research Product CycLex TrkA Kinase Assay Inhibitor Screening Kit is designed to measure the activities of recombinant catalytic domain of TrkA for the rapid and sensitive evaluation of inhibitors or activators The phosphotyrosineySpecific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in recombinant Tyrosine kinase substrate 1 which is efficiently phosphorylat
15. rder 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 immediately after wash Reagent Stability All of the reagents includedin the CycLex Research Product CycLex TrkA Kinase Assay Inhibitor Screening Kit have been testedifor stability Reagents should not be used beyond the stated expiration date Upon receipt kit re gents should be stored at 4 C Coated assay plates should be stored in the original foil bag sealed by the yzip lock and containing a desiccant pack For research us only not for use in diagnostic or therapeutic procedures Cat CY 1091 9 Version 140318 4 TrkA Kinase Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant TrkA catalytic domain enzyme reaction 0 1 2 3 4
16. rt half life It is particularly unsuitable when kinase assays are only performed on an infrequent basis The CycLex Research Product CycLex TrkA Kinase ASsay Imhibitor 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 TrkA catalytic domain Cat CY 1091 2 Version 140318 TrkA 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 TrkA Kinase Assay Inhibitor Screening Kit is a single site semi quantitative immunoassay for kinase activity of recombinant catalytic domain of TrkA Plates are pre coated with a newly designed Tyrosine kinase substrate 1 which can be easily phosphorylatedyby recombinant catalytic domain of TrkA The detector antibody is PY 39 an antibody that specifically detects the phosphotyrosine residue on Tyrosine kinase substrate 1 The CycLex Research Product CycLex TrkA Kinase Assay Inhibitor Screening Kit might be used to follow the kinetics of recombinant catalytic domain of TrkA as well as screening TrkA intibitor or activator To perform the test the recombinant catalytic domain of TrkA is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate Tyrosi
17. 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 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 1091 5 Version 140318 TrkA 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
18. ve Control to each well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 C for 30 minutes 2 Follow the StandardiAssay steps 5 10 page 6 7 Cat CY 1091 Version 140318 TrkA 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 TrkA sample duplicates Positive Control and all experimental sample duplicate values when applicable When the TrkA Positive Control 1 unit assay is included as an internal control for the phosphorylation reaction the absorbance value should be greater than 2 with a background less than 0 15 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 TrkA Kinase Assay Inhibitor Sereening Kit has been shown to detect the kinase activity of recombinant catalytic domain of TrkA dhe assay shows good linearity of sample response Troubleshooting 1 The TrkA 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 o

CY-1091

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