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CY-1166

1. e The buffers and reagents in this kit may contain preservatives or othemchemi als 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 or in argas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containifg solutions in compliance with local regulations e Avoid contact with Substrate Solution which contains hydrogen peroxide e Avoid contact with Stop Solution which contains Sulfuric Acid e In case of contact with the Stop Solutiongandth Substrate Solution wash skin thoroughly with water and seek medical attention when necessary e Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols W ar protective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1166 5 Version 141028 oe MAPKAP kinase 2 Assay Inhibitor Screening Kit aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CycLex MAPKAP kinase 2 Assay Inhibitor Screening Kitais 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 Since
2. R I and Huang C K Bl100d187 5287 5296 1996 8 Lee J C Laydon J T McDonnell P C Gallagher T F Kumar S Green D McNulty D Blumenthal M J Heys R R Landvatter S W Strickler J E McLatighlin M M Siemens I R Fisher S M Livi G P White J R Adams J L and Young P R Nature 372 739 746 1994 9 Xia Z Dickens M Raingeand J Davis R J and Greenberg MZ Seience 270 1326 1331 1995 10 Saklatvala J Rawlinson L Waller R J Barnes M J and Farndale R W J Biol Chem 271 6586 6589 1996 11 Zu Y L Ai Y and Huang C K J Biol Chem 270 202 206 1995 12 Engel K Schultz H Martin F Kotlyaroo A Plath K Hahn M Heinemann V and Gaestel M J Biol Chem 270 27213 27331 1995 13 Ben Levy R Leighton I A Doza Y N Attwood P Morrice N Marshall C J and Cohen P EMBO J 14 5920 5930 1995 14 Huang CK Zhan L Ai Y Jongstra J J Biol Chem 272 17 9 1997 15 Hannigan M Zhan L Ai Y Kotlyarov A Gaestel M and Huang C K 2001 J Immunol 167 3953 3961 Related Products MAPKAP kinase 2 Positive control Cat CY E1167 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 wwyney clex co ip CycLex Circulsex products are supplied for research use only CycLex CircuLex products and components thereof may not b
3. is in the sample the high level of A450 is not observed in Inhibitor control ATP minus contr6l and No enzyme control Inhibitor ATP minus Positive N e zyme Assay reagents Test Sample control control control control Kinase Reaction Buffer 90 pL 80 uL 90 pL 90 uL Kinase Buffer provided 90 uL 10X Staurosporine 500 uM 10 uL z Your enzyme fraction 10 uL 10 uL 10 uL MAPKAP kinase 2 Positive g g _ 10 uL _ Control 2 m unit uL u Buffer 10 uL 10X Staurosporine 500 uM See page 4 section Materials Required but nof Provided Cat CY E1166 See page 4 section Materials Required but not Provided 1 Following the above table add the Reagents to each well of tthe microplate Finally initiate the reaction by adding 10 uL of Your enzyme fraction or Buffer t each well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 C for 30 60 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Evaluation of Results 1 Average the absorbance values for the MAPKAP kinase 2 sample duplicates positive control and all experimental sample duplicate values when applicable When the MAPKAP kinase 2 positive control 20 m units assay is included as an internal control for the phosphorylation reaction the absorbance value should be greater than l 0 With a background less than 0 15 2 Fo
4. 15 30 45 60 75 90 105 120 Q Reaction Time min C CY 1166 11 Version 141028 Fae MAPKAP kinase 2 Assay Inhibitor Screening Kit YCLEX User s Manual A For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of broad protein kinase inhibitor Staurosporine on activity of recombinant MAPKAP kin o 7 8 S 3 8 B C Relative intensity of control 1 0 10 0 100 0 1000 0 10000 0 100000 0 S So Staurosporine nM C CY 1166 12 Version 141028 oe MAPKAP kinase 2 Assay Inhibitor Screening Kit cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References Stokoe D Campbell D G Nakielny S Hidaka H Leevers S J Marshall C and CohengaP EMBO J 11 3985 3994 1992 2 Rouse J Cohen P Trigon S Morange M Alonso Liamazares A Zamannillo D Hunt T and Nebreda A R Cell 78 1027 1037 1994 3 Freshney N W Rawlinson L Guesdon F Jones E Cawley S Hsuan J and Saklatvala J Cell 78 1039 1049 1994 4 Han J Lee J D Bibbs L and Ulevitch R J Science 265 808 811 1994 5 Galcheva Gorgova Z D rijard B Wu I H and Davis R J Science 265 806 808 1994 6 Kotlyarov A Neininger A Schubert C Eckert R Birchmeier C Volk H D a d Gaestel M Nat Cell Biol 1 94 97 1999 7 Zu Y L Ai Y Gilchrist A Labadia M E Sha afi
5. 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 supplied and are sufficient for the one 96 well 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 full length LSP1 as substrate of MAPKAP kinase 2 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 for Kinase Reaction Buffer and sample dilution 20X ATP One vial of lyophilized ATP Naz salt HRP conjugated Detection Antibody One vial containing 12 mL of RP horseradish peroxidase conjugated anti phospho LSP1 S204 AT 1E6 antibody Ready to usea 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 H2SOx Ready to lise Materials Required but not Provided e MAPKAP kinase 2 positive control Available from CycLex Cat CY E1166 One vial contains 4 units 100 uL MAPKAP kinase 2 enzyme Positive control should be added to the first well at 20 m units well For instance diluted positivescontrol 1 20 use 10 uL for 1 assay Unused MAPKAP kinase 2 enzyme should be stored in aliquots at 70 C e 10X Staurosporine 500 u
6. M Staurosporin is available from Sigma Cat S 4400 50 mM stock solution DMSO diluted 1 100 in Kinase Buffer e Pipettors 2 20 uL 20 200 uL and 200 1000 pL precision pipettors with disposable tips e 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 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 of 450 nm which will give a somewhat higher reading 500 or 1000 mL graduated ylinder e Reagent reservoirs Deionized watenrof the highest quality Disposable paper towels Cat CY 1166 4 Version 141028 oe MAPKAP kinase 2 Assay Inhibitor Screening Kit aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption 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 e Use deionized water of the highest quality e Do not mix reagents from different kits
7. anti phospho LSP1 serine204 monoclonal antibody as a reporter molecule in a 96 well ELISA format This assay provides a non isotopic sensitive and specific method to detect MAPKAP kinase 2 activity Cat CY 1166 2 Version 141028 oe MAPKAP kinase 2 Assay Inhibitor Screening Kit cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex MAPKAP kinase 2 Assay Inhibitor Screening Kit ispa single site semi quantitative immunoassay for MAPKAP kinase 2 activity Plates are pre coated witha substrate corresponding to recombinant LSP1 Leukocyte Specific Protein which contains_a Serine residue that are phosphorylated by MAPKAP kinase 2 MAPK activated protein kinase 2 The detector antibody specifically detects only the phosphorylated form of serine 204 lon LSP1 The CycLex Research Product CyecLex MAPKAP kinase 2 Assay Inhibitor Screening Kit can be used to study the kinetics of a purified or partially purified MAPKAP kinase 2 as well as to screening these kinases inhibitor To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substrate in the presence of Mg and ADP The amount of phosphorylated substrate is measured by binding it with a horseradish peroxidase conjugate of AT 1E6 an anti phospho LSP1 serine 204 specific antibody which then catalyzes dite cofiversion of the chromogen
8. ate 3 Duplicate wells containing 20 m units 10 uL MAPKAP kinase 2 Cat CY E1166 should be included in each assay as a positive control for phosphorylation 4 Begin the kifaselreaft n by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer and incubate 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 of HRP conjugated Detection Antibody into each well cover with a plate sealer andincubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate Cat CY 1166 6 Version 141028 SR MAPKAP kinase 2 Assay Inhibitor Screening Kit f 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 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 Readithe plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes f adding th
9. e Stop Solution 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 pouch 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 To assay individual column fractions add 10 uL of each fraction to the wells of the assay plate on ice Duplicate wells containing 20 m units 10 uL MAPKAP kinase 2 Cat CY E1166 should be included in each assay as a positive control for phosphorylation 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 pt 8 0 to ach well 6 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle dapping or aspiration 7 Pipette 100 uL of 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 after use oo Wash wells five times with Wash Buffer making sure each well is filled completely R
10. e 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 1166 13 Version 141028
11. emove 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 10 15 minutes 10 Adh 100ML 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 dual wavelengths of Cat CY 1166 7 Version 141028 my MAPKAP kinase 2 Assay Inhibitor Screening Kit Pa c ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 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 minutes of adding the Stop Solution Recommendations Special considerations when screening activators or inhibitors In order to estimate the inhibitory effect on MAPKAP kinase 2 activity in the test chemicals correctly it is necessary to conduct the control experiment of Solvent control at least once for every 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 cause an inhibitory effect on MAPKAP kinas gt 2 activity the level of A450 is weakened as compared with Solvent control The high level of A450 i not observed in Inhibitor control usually A450 lt 0 2 Assay reagent
12. experimental conditions may vary an aliquot of the MAPKAP kinase 2 Cat CY E1166 available separately from CycL x shouldbe included in each assay as a positive control 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 ddH20 Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare 20X ATP Solution by adding 0 8 mL of ddH20 to the viakof 20X ATP provided lyophilized Mix gently until dissolved the Final concentration of the 20X ATP Solution should be 2 5 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 uL 100 uL You will need 80 90 uL of Kinase Reaction Buff mper 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 wellsto the foil pouch refold seal with tape and store at 4 C 2 Prepare all samples diluted with Kinase Buffer as needed All samples should be assayed in duplic
13. f this kit include 1 Screening inhibitors or activatogs of MAPKAP kinase 2 2 Detecting the effects of pharmacological agents on MAPKAP kinase 2 activity This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all components at 4 C e Don t expose reagents to excessive light Cat CY 1166 1 Version 141028 MAPKAP kinase 2 Assay Inhibitor Screening Kit jde bers Mi GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures Introduction IAP kinase activated protein kinase 2 MAPKAP kinase 2 was originally identified as a substrategfor the p42 p44 MAPKs in vitro However recent data indicate that in intact cells the upstream kinase that regulates MAPKAP kinase 2 is p38 MAPK Treatment of cells with endotoxin interleukin 1 tumor necrosis factor or various stress stimuli activate p38 MAPK and MAPKAP kinase 2 Mice deficient in MAPKAP kinase 2 showed a reduction in bacterial lipopolysaccharide induced biosynthesis of tumor necrosis factor TNF interferon interleukin IL 1 IL 6 and nitric oxide Suggesting a critical role of MAPKAP kinase 2 in inflammatory cytokine production In human neutrophils MAPKAP kinase 2 is also activated by the chemotactic factor f Met Leu Phe and phorbol 12 myristate 13 acetate The function of MAPKAP kinase 2 is not known but its upstream kin s p38 MAPK has been proposed
14. ic 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 reflects the relative amount of MAPKAP kinase 2 activity in the sample For kin tic analysis the sample containing MAPKAP kinase 2 is added to the wells in a similar fashion and at varying times the reaction is stopped by the addition of a chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product CycLex MAPKAP kinase 2 Assay Inhibitor Screening Kit is designed to accurately determine the presence and relative amount of MAPK AP kinase 2 kinase activity in purification column fractions and to determine non isotopic Kinetic analysis of MAPKAP kinase 2 activity Careful attention to methods of chromatography and the assay protocol will provide the investigator with a reliable tool for the evaluation of MAPKAP Kinase 2 activity Summary of Procedure Add 100 uL of sample to the wells 4 Incubate for 30 min at 30 C Wash the wells t Add 100 uIORHRP conjugated anti phosphorylated form specific antibody g Incubate for 60 min at room temp Wash the wells t Ad amp 100 uL of Substrate Reagent t Add 100 uL of Stop Solution t Measure absorbance at 450 nm Cat CY 1166 3 Version 141028 oe MAPKAP kinase 2 Assay Inhibitor Screening Kit aa ycLex User s Manual
15. ods 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 desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Sabstrate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Research Product ycLex MAPKAP kinase 2 Assay Inhibitor Screening Kit have been tested for stabilityAReagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at4 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Cat CY 1166 10 Version 141028 Pae MAPKAP kinase 2 Assay Inhibitor Screening Kit e NycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant MAPKAP kinase 2 enzyme reaction 25 7 2 0 F A450 0 5 F 0 0 bt sill Pi iiiiuil ot til 144 iiiul Looi 0 0 0 1 1 0 10 0 100 0 1000 0 MAPKAP kinase 2 mU Fig 2 Time course of recombinant anaran V 25 p A450 0
16. r screening of purification chromatography fractions on graph paper plot the mean absorbance values for each of the samples onthe Y axis versus the fraction number on the X axis to determine the location of the eluted purified MAPKAP kinase 2 3 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 Res atch Product CycLex MAPKAP kinase 2 Assay Inhibitor Screening Kit has been shown to deteet thepactivity of MAPKAP kinase 2 in column fractions of human or animal cell extracts The assay shows good linearity of sample response The assay may be used to follow the purification of MAPKAP kinasel2 Cat CY 1166 9 Version 141028 oy MAPKAP kinase 2 Assay Inhibitor Screening Kit P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The MAPKAP kinase 2 positive control should be run in duplicate using the protocol described ingthe 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 as can assay kinetics that are other than first order For a non linear curve point to point or quadratic curve fit meth
17. ry MAPKAP kinase 2 Assay Inhibitor Screening Kit U kd a ycLex ser s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring MAPKAP kinase 2 activity CycLex MAPKAP kinase 2 Assay Inhibitor Screening Kit Cat CY 1166 Intended Use ccccccccccccceeceeeeeceeeeeeees 1 Be sat nsatsinanigayonmnysaneiocsiciassadsaepiaaeeeisinen 1 Tntroduction cceceeeecceecseeesssseesessesenseees 2 Principle Of the Assay 3 Materials Provided ceccceeeseeeeeeeeeeees 4 Materials Required but not Provided 4 Precautions and Recommendation 5 Detailed Protocol cecceccceeesseseseeeeeees 6 8 Evaluation of Results cccccceeeeeeeeeeeee 9 Assay Characteristics seee 9 TroubleshOOtinig s ssinsseosssssantsondasneasssnsnasivans 10 Reagent Stability 10 Example of Test Results eee 11612 RefCLENCES cccdcisssissssasseiagsessebadsiaacastecneeereoae 13 Related Products ccccccccccccccceeeeeeeeeeees 13 Intended Use The CycLex Research Product CycLex MAPKAP kinase 2 Assay Inhibitor Screening Kit is designed to measure the activities of purified MAPKAP kinase 2 for the rapid and sensitive evaluation of inhibitors or activators The phospho serin specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phospho serine204 residue in LSP1 which is phosphorylated by MAPKAP kinase 2 Applications o
18. s Test Solvent Inhibitor sample control control Kinase Reaction Buffer 80 uL 80 uL 80 uL 10X Inhibitor or equivalent 10 nL Solvent for Inhibitor 10 pL 10X Staurosporine 500 uM 10 uL MAPKAP kinase 2 Positive Control 2 m unit uL 10 uL 10 uL 10 uL or your enzyme fraction 10X Staurosporine 500 uM See page 4 section Materials Required but not Provided Cat CY E1166 See page 4 section Materials Required but not Provided 1 Following the above table add the Reagents to eachwell of the microplate Finally initiate reaction by adding 10 uL of Diluted MAPKAP kinase 2 positive control to each well and mixing thoroughly at room temperature Cover with plate sealer incubate at 30 C for 30 60 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Cat CY 1166 8 Version 141028 y AA MAPKAP kinase 2 Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures Special considerations when measuring precise MAPKAP kinase 2 activity In order to measure the activity of MAPKAP kinase 2 correctly it is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and ATP minus control at least once for the first experiment in addition to No enzyme control as indicated in the following table Although the level of A450 increases in Test sample when MAPKAP kinase 2 enzyme activity
19. to be involved in the biosynthesis of inflammatory cytokines apoptosis and platelet aggregation MAPKAP kinase 2 contains a C terminal autoinhibitory domaing gand is activated by phosphorylation at multiple sites gt Recently It was reported that the major substrate for MAPKAP kinase 2 in human neutrophils is not HSP27 but is a protein termed p60 which we was identified as LSP1 4339 amino acid cytoskeletal protein the expression of which is restricted to neutrophils lymphocytes and macrophages Measurement of MAPKAP kinase 2 activity The protocol generally regarded as most sensitive for the quantitative measurement of MAPKAP kinase 2 activity involves incubation of the MAPKAP kinase 2 sample with substrate either a natural or synthetic polypeptide such as Long S6 Kinase substrate peptide in the presence of Mg 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 MAPKAP kinase 2 Assay Inhibitor Screening Kit uses a peroxidase coupled

CY-1166

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