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CY-1162

1. 1 Screening inhibitors or activators of checkpoint kinases 2 Detecting the effects of pharmacological agents on checkpoint kinases This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt store all components at 4 C Don t exposefredgents to excessive light Cat CY 1162 1 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Three different human Cdc25 family members exist with Cdc25A regulating the G1 S transition and Cdc25B and Cdc25C involved in G2 M progression Evidence suggests that two critical amino acids threonine 14 and tyrosine 15 located within the cyclin dependent kinases represent the major target for the Cdc25 family of protein phosphatases Dephosphorylation of these two critical amino acid residues ts essential for proper cell cycle progression and the subsequent association of cyclin dependent kinases with their associated cyclins 1 Given their crucial role in cell cycle progression and checkpoint control the regulation of th activity of the various Cdc25 family members has been the subject of numerous investigations For the case of Cdc25C enzymatic activity has been demonstrated to be low during interphase dn part because the phosphatase is phosphorylated on serine 216 In response to DNA damage various intfac llular
2. 2 5000 R 0 996 4 000 23000 A 2 000 0 0 20 40 60 80 100 S CHE C CY 1162 14 Versions 140318 FS Checkpoint Kinase Assay Inhibitor Screening Kit 1 es vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 3 Km for ATP recombinant C TAK1 6 000 p y 53 501x 20 514 5 000 R 0 9999 N 4000 F Z 3 000 2 000 1 000 0 0 20 40 60 80 100 S Fig 4 1 Effect of broad spectrum kinase inhibitor M activity fi 120 0 100 0 z S o 80 0 D a 60 0 5 7 40 0 o 2 200 c 0 0 0 20 40 60 80 100 Staurosporine conc nM C CY 1162 15 Versions 140318 P Checkpoint Kinase Assay Inhibitor Screening Kit 1 t y ex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 4 2 Effect of broad spectrum kinase inhibitor staurosporine on Chk2 activity EN t09 100 0 E E g 89r 800 eo l amp 600 a 2 E a 2 40 2 40 0 E o E 20 L amp 20 0 2 A 0 0 0 5 10 15 20 0 0 25 0 5 0 75 1 Staurosporine conc uM Staurosporine conc uM Fig 4 3 Effect of broad spectrum kinase inhibitor staurosporine T activity 120 S 100 o o re 80 En a 60 m E 40 o amp s 20 o 0 0 20 40 60 80 100 Staurosporine conc nM C CY 1162 16 Versions 140318 Checkpoint Kinase Assay Inhibitor Scr
3. S 4400 See Page 4 section Materials Required but not Provide Chk1 positive control Cat CY E1162 1 Chk2 positive control atit E1162 2 and C TAKI Cat CY E1162 3 See Page 4 section Materials Required but not Provided 1 Following the above table add the Reagents to by adding 10 uL of CycLex checkpoint kina temperature Cover with plate sealer or lid and incub ell of the microplate Finally initiate reaction to each well and mixing thoroughly at room t 30 C for 60 minutes 2 Follow the Standard Assay steps 5 10 pag 167 C CY 1162 8 Versions 140318 P Checkpoint Kinase Assay Inhibitor Screening Kit 1 t Vclex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Special considerations when measuring precise Checkpoint kinase activity In order to measure the activity of checkpoint kinase family correctly it is necessary to conduct t 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 followin Although the level of A450 increases in Test sample when checkpoint kinase family enzyme acti is in the sample the high level of A450 is not observed in Inhibitor control ATP minus i No enzyme control Assay reasenis Test Inhibitor ATP minus Positi enzyme y reag Sample control control control control Kinase React
4. on graph paper plot the me n absorbance values for each of the samples on the Y axis versus the fraction number on the X axis to determine the location of the eluted purified individual checkpoint kinase 3 For kinetic analysis on graph paper plot the mean absorbance values for each of tlie time points on the Y axis versus the time of each reaction minutes on the X axis Assay Characteristics The CycLex Research Product Checkpoint Kinase Assay Inhibitor Sereening Kit 1 has been shown to detect the activity of indicated checkpoint kinases in the column fractions of mammalian cell lysates The assay may be used to follow the purification of individual checkpoint kinase Troubleshooting 1 The CycLex checkpoint kinase should be run in duplicate when a standard assay is being performed using the 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 kineties of the assay is of the first order Variations in the protocol can lead to non linearity of the curve as can assay kinetics of other than first order For a non linear curve point to point or quadraticcurve fit methods should be used 3 Poor duplicates accompanied by elevatedpvalu s for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately su
5. to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled Dispose of tetra methylbenzidine TMB containing sol tions in compliance with local regulations Avoid contact with Substrate Solution which contains hydrogen peroxide Avoid contact with Stop Solution which contains Sulfuric Acid In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention whenNecessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1162 5 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Checkpoint Kinase Assay Inhibitor Screening Kit 1 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 Since conditions may vary running an aliquot Of the appropriate checkpoint kinase positive control Cat CY E1162 1 3 separately av
6. kinases including Chk1 Chk2 and C TAK1 appear to phosphorylate Cdc25C on this residue 226 One of the important functional consequences of phosphorylation of Ser 216 1s fo create a consensus binding site for 14 3 3 protein binding 4 A variety of evidence suggests that in human cells the binding of 14 3 3 increases the cytoplasmic localization of the proteing 7 9 In addition to 14 3 3 binding Cdc25C is also actively transported from the nucleus through a l ptomyein B sensitive pathway that requires an N terminal nuclear export sequence 9 Measurement of checkpoint kinases Chk1 Chk2 and TAKT activity The protocol generally regarded as most sensitive for the quanttative measurement of checkpoint kinase activity involves incubation of the checkpoint kinase Sample with substrate either a natural or synthetic polypeptide such as Chktide substrate peptide 4mthe 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 counted While sensitive this method is labor intensive generates hazardous radioactive waste and depends on radioisotope of short half life It is particularly unsuitable when kinase assays are only performed on an infrequent basis The CycLex Checkpoint Kinase Assay Inhibitor Sc
7. 216 specific antibody which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution to a blue solution or yellow after the addition of stopping reagent The color is quantitated by spectrophotometry and reflects the relative amount of checkpoint Kinases activity in the sample For kinetic analysis the checkpoint kinase containing sample is added to the wells in a similar fashion and at varying times the reaction is stopped by the additionof the chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before Summary of Procedure Add 100 uL of sample to the wells 1 Incubatedfor I hr at 30 C Wash the wells Add 100 uL of HRP conjugated anti phospho specific antibody 1 Incubate for 1hr at room temp Wash thegwells y Add 100 uL of Substrate Reagent Add 001 of Stop Solution t Measure absorbance at 450 nm Cat CY 1162 3 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex 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 suppliedand 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 aforl zip lock bag with a desiccant pack Wells are coated with recombinant Cd
8. Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring Chk1 Chk2 and C TAK1 Activities CycLex Checkpoint Kinase Assay Inhibitof Screening Kit Cat CY 1162 Intended Use ee 1 wire n N RM cT rS 1 Introduction sss sisirin 2 Principle of the Assay 3 Materials Provided sss 4 Materials Required but not Provided 4 Precautions and Recommendations 5 Detailed Protocol esses 6 9 Evaluation of Results ss 10 Assay Characteristics s 10 Troubleshooting si sieiawstussnccatesnrieieaasssunesensons 10 Reagent Stability ieeon ipo E tkt ino tpa RES 10 Example of Test Results one 14616 References eror aiana 17 Related Products cccccccccecceesseesseeeeeees 17 Intended Use The CycLex Research Product CycLex Checkpoint Kinase Assay Inhibitor Screening Kit 1 is designed to measure the activities of pufified checkpoint kinase enzyme such as Chk1 Chk2 and C TAKI for the rapid and sensitive evaluation of checkpoint kinase inhibitors using recombinant checkpoint kinases The phospho specifie monoclonal antibody used in this assay kit has been demonstrated to recognize the phoSphosserine 216 residue in Cdc25C which is phosphorylated by checkpoint kinases Applications of this kit include
9. Checkpoint Kinase Assay Inhibitor Screening Kit 1 es vyclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 1 3 Dose dependency of recombinant C TAK1 enzyme reaction XN 2 0 1 5 2 L0 lt 0 5 0 0 0 5 10 15 20 25 30 35 C TAK dose mUnit wv Fig 2 1 Time course of recombinant Chk1 enzyme e A 1 25 gt 1 00 0 75 A450 0 50 0 25 0 00 0 15 30 45 60 75 90 Reaction Time min C CY 1162 12 Version 140318 FS Checkpoint Kinase Assay Inhibitor Screening Kit 1 es yclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 2 2 Time course of recombinant Chk2 enzyme reaction A450 0 15 30 45 60 75 90 Reaction time min Y Fig 2 3 Time course of recombinant C TAK1 E oa 1 50 gt 1 25 1 00 0 75 A450 0 50 0 25 0 00 0 15 30 45 60 75 90 Reaction time min C CY 1162 13 Versions 140318 FS Checkpoint Kinase Assay Inhibitor Screening Kit 1 es vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 1 Km for ATP recombinant Chk1 2 2500 R 0 9981 2 000 3 000 y 51 643x 70 388 i e 1 00 1 000 500 S V Km for ATP 1 36uM 0 10 20 30 40 50 S Fig 3 2 Km for ATP recombinant Chk2 Ww 6000 gt y 46 186x 153 2
10. ailable from CycLexy should be included in each assay Disposable pipette tips and reagent troughs should be used for all 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 ddH5O Mix well Store at 4 C for two weeks or 20 C for long temmstorage 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 the 20X A TP 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 10mL 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 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 2 Prepare all samples diluted with Kinase Buffer as needed w Duplicate wells containing 10 E of checkpoint kinase positive control 10 m units should be included in each assay ds a positive control for phosphorylatio
11. c25C as Checkpoint kinases substrate 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 Na salt HRP conjugated Detection Antibody One vial containing 20 mL of HRP h rseradish peroxidase conjugated anti phospho Cdc25C S216 1F1 antibody 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 H5SO Ready to use Materials Required but not Provided Recombinant Chk1 Chk2 and C TAKI positive control Available from CycLex Chk1 positive control Cat CY E1162 1 Chk2 positive control Cat CY E1162 2 and C TAKI Cat CY E1162 3 One vial containing 2 Units checkpoint kinase enzyme Positive control should be added to the first well at 10 m units well For instance diluted positive control 1 m unit uL use 10 uL for 1 assay Unused checkpoint kinase enzyme should be stored in aliquots at below 70 C 10X Staurosporine 10 uM Staurosporin is available from Sigma Cat S 4400 1 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 Precision repeating pipettor e Wash bottle or multichannel dispenser for plate washing M
12. ch results indicate a need for washer maintenance 4 Overall low signal may indicatethat desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Research Product CycLex Checkpoint Kinase Assay Inhibitor Screening Kit Ighave been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the ATP and checkpoint kinase component mu t be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zipdock and containing a desiccant pack For xesearch use only not for use in diagnostic or therapeutic procedures Cat CY 1162 10 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 c Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 1 Dose dependency of recombinant Chk1 enzyme reaction 2 0 1 3 e ATP 1 0 o ATP A450 0 5 0 0 0 10 20 30 40 Chk1 dose mUnit Fig 1 2 Dose dependency of recombinant Chk2 enzyme reaction 1 50 1 00 c uv e ATP 0 50 e ATP 0 00 0 5 10 15 20 Chk2 dose mUnit Cat CY 1162 11 Version 140318 FS
13. eening Kit 1 4 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Obaya A J and Sedivy J M Cell Mol Life Sci 59 126 142 2002 2 Walworth N C Davey S and Beach D Nature 363 368 371 1993 3 Walworth N and Bernards R Science 271 353 356 1996 4 Peng C Y Graves P R Thoma R S Wu Z Shaw A S and Piwnica Worms H Science 277 1501 1505 1997 5 Furnari B Rhind N and Russell P Science 277 1495 1497 1997 6 Sanchez Y Wong C Thoma R S Richman R Wu Z Piwnica Worms H and Elledge S J Science 277 1497 501 1997 7 Dalal S N Schweitzer C M Gan J and DeCaprio J Mol Cell Biol 19 4465 4479 1999 8 Graves P R Yu L Schwartz J K Sausville E A O Conner P M and Piwnica Worms H J Biol Chem 2775 5600 5605 2000 9 Graves P R Lovly C M Uy G L and Piwnica Worms H Oncogene 20 1839 1851 2000 Related Products Chk1 Positive control Cat CY E1162 1 Chk2 Positive control Cat CY E1162 2 C TAKI Positive control Cat CY E1162 3 Anti phospho Cdc25C Ser216 monoclonal antibody 1F1 Cat CY M1018 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 481 265 7647618 e mail info cyeclex co jp URL http wwweeyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and compo
14. he previously added Substrate Reagent 11 Measure absorbance in each well using a spectorphotometric plate reader at dual wavelengths of 450 5409 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 Cat CY 1162 T Version 140318 P Checkpoint Kinase Assay Inhibitor Screening Kit 1 t y ex User s Manual For Research Use Only Not for use in diagnostic procedures Stop Solution Recommendations Special considerations when screening activators and inhibitors In order to estimate the inhibitory effect on individual checkpoint kinase activity in the correctly it is necessary to conduct the control experiment of Solvent control at least experiment and Inhibitor control at least once for the first experiment in addition to Te indicated in the following table When test chemicals cause an inhibitory effect on individual kinase activity the level of A450 is weakened as compared with Solvent control The high level of A450 is not observed in Inhibitor control usually A450 0 3 eA Solv ibitor Assay reagents Test sample co vonteol Kinase Reaction buffer 80 uL 80 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10X Staurosporine 10 uM 10 uL CycLex checkpoint kinase 1 m unit uL 10 10 uL 10 uL or your enzyme fraction Cat
15. icrocentrifuge and tubes for sample preparation Vortex mixer 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 cylinder Reagent reservoirs Deionized water f the highest quality Disposable paper towels Cat CY 1162 4 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Store the checkpoint kinase enzyme at below 70 C and the ATP at 20 C when not in use Storegall other components at 4 C Do not expose reagents to excessive light Avoid freeze thaw cycles Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock peuch Which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residue from glassware Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or other chemicals Care should be taken
16. ion buffer 90 uL 80 uL 90 uL Kinase Buffer provided 90 uL 10X Staurosporine 10 uM 10 uL Your enzyme fraction 10 uL 10 uL 10 CycLex checkpoint kinase 1 m unit uL 10 uL Buffer 10 pL Cat S 4400 See Page 4 section Materials Required but not Provided Chk1 positive control Cat CY E1162 1 Chk2 positive control Cat CY E See Page 4 section Materials Required but not Provided and C TAKI Cat CY E1162 3 1 Following the above table add the Reagents to each well reaction by adding 10 uL of Your enzyme fraction or at room temperature Cover with plate sealer or lid and i 2 Follow the Standard Assay steps 5 10 page Q Q N amp Y 2 roplate Finally initiate the each well and mixing thoroughly t 30 C for 60 minutes C CY 1162 9 Versions 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Average the absorbance values for the checkpoint kinase sample duplicates positive control andgall experimental sample duplicate values when applicable When checkpoint kinase positive control 10 m units assay is included as an internal control for the phosphorylation reaction the absorbance value should be greater than 1 0 with a background less than 0 15 2 For screening of purification chromatography fractions
17. n 4 Begin the kinase reactionsby addition of 90 uL Kinase Reaction Buffer per well cover with plate sealer or lid and incubate at 30 C for 60 minutes Nn 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 1004i1L of HRP conjugated Detection Antibody into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate after s Cat CY 1162 6 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex 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 Remove 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 Re d tbe plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutesgof adding the Stop Solution Kinetic Assays 1 Remove the appropriate number of microtiter wells from the foil pouch and place them i
18. nents 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 1162 17 Version 140318
19. nto the well holder Return any unused wells to the foil pouch refold seal with tap and store at 4 C 2 Prepare all samples diluted with Kinase Buffer as needed 3 Duplicate wells containing 10 uL of checkpoint kinase positive control 10 m units should be included in each assay as a positive control for phosphorylation 4 Begin the kinase reaction by addition of 90 wl 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 cover with plate sealer or lid and 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 ach well 6 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 7 Pipette 100 uL of HRP conjugated Detection Antibody into each well cover with plate sealer or lid 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 residual Wash Buffer By gentle tapping or aspiration 9 Add 100 uL of S bstrate 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 t
20. reening Kit L uses a peroxidase coupled anti phospho Cdc25C S216 monoclonal antibody as a reporter molecule 4n ja 96 well ELISA format This assay provides a non isotopic sensitive and specific method measure the activities of checkpoint kinases Cat CY 1162 2 Version 140318 Checkpoint Kinase Assay Inhibitor Screening Kit 1 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Products CycLex Checkpoint Kinase Assay Inhibitor Screening Kit 1as a single site semi quantitative immunoassay for checkpoint kinase activity Plates are pre coated witha substrate corresponding to recombinant Cdc25C which contains serine residues that cam be phosphorylated by checkpoint kinases including Chk1 Chk2 and C TAKI The detector antibody specifically detects only the phosphorylated form of serine 216 residue on Cdc25C The CycLex Checkpoint Kinase Assay Inhibitor Screening Kit 1 may be used to study the kinetics of a purified or partially purified individual checkpoint kinase Chk1 Chk2 and C TAK1 as well as to screening individual checkpoint kinase inhibitor To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound sub trate following the addition of Mg and ATP The amount of phosphorylated substrate is measured by binding it with a horseradish peroxidase conjugate of 1F1 a anti phospho Cdc25C serine

CY-1162

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