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CY-1179

1. Noyenzyme y reag Sample control control control control Kinase Reaction Buffer 90 uL 80 uL 90 uE 90 uL Kinase Buffer provided 90 uL 10X Staurosporin 10 uM 10 pL lt Your enzyme fraction 10 uL 10 uL 10 uL Human Mps1 Positive Control _ 10 uL _ 2 m unit uL Buffer for Your enzyme fraction 10 uL 10X Staurosporin See page 4 section Materials Required but not Provided Cat CY E1179 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 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 1179 Version 140318 Human Mps 1 TTK 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 human Mps1 TTK positive control duplicates and all experimental sample duplicate values when applicable When the human Mps1 positive control 10 m units assay is included as an internal control for the phosphorylation reaction the absorbance y lue should be greater than 1 0 with a background less than 0 15 2 For screening of purification chromatography fr
2. holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C Prepare all samples diluted with Kinase Buffer as needed All samples should be assayed in duplicate To assay partially purified re ombinant human Mps1 add 10 uL of each fraction to the wells of the assay plate on ice Duplicate wells containing 20 m units 10 uL human Mps1 TTK positive control Cat CY E1179 should be included in each assay as a positive control for phosphorylation Begin kinase reaction bysaddition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervals suggest dimterval is 5 minutes but should be individually determined for each system After the final addition incubate at 30 C for 20 minutes 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 Wash wellsifive times with Wash Buffer making sure each well is filled completely Remove fesidual Wash Buffer by gentle tapping or aspiration Pipett 100 uL of HRP conjugated Detection Antibody into each well cover with a plate sealer Catt CY 1179 7 Version 140318 yclex Human Mps 1 TTK kinase Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate 8 Wash wells five times as sam
3. A R Meehl J B Morgan G j Schutz Geschwender A and Winey M The yeast protein kinase MpsIp is required for assembly of the integral spindle pole body component Spc42p J Cell Biol 156 453 465 2002 15 Winey M Huneycutt BJ Gentrosomes and checkpoints the MPS1 family of kinases Oncogene 21 40 616 149 2002 Cat CY 1179 13 Version 140318 Fag Human Mps1 TTK kinase Assay Inhibitor Screening Kit a ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products Human Mps1 Positive control Cat CY E1179 amp Y Oo 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 cyclex co jp CycLex CircuLex products are supplied esearch use only CycLex CircuLex products and components thereof may not be resol for resale or used to manufacture commercial fr a products without prior written appro CycLex Co Ltd To inquire about licensing for such commercial use please contact_us il amp Y amp Y C CY 1179 14 Version 140318
4. Solution Cat CY 1179 5 Version 140318 Human Mps 1 TTK 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 Human Mps1 TTK kinase Assay Inhibitor Screeningdgit 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 experimental conditions maay vary an aliquot of the human Mps1 TTK positive control Cat CY E1179 available separately from CycLex should be 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 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 0 8 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 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 pL 20X ATP Solution 0 5 mL 5
5. any unused conjugate Wash wells five times as same as in step 5 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 5 15 minutes 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 dualjwavelengths of 450 540 nm Dual wavelengths 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 Note 1 Complete removal of liquid at each step is essential to good perfoffmanc After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the 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 human Mps1 TTK p6sitive control Note 3 If the microplate reader is not capable of reading abSoxbane e greater than the absorbance of the Mps1 TTK positive control perform a second reading at 405 nm A new O D values measured at 405 nm is used to determineghumam Mps activity of off scale samples The readings at 405 nm should not replace the on seale readings at 450 nm Kinetic Assay 1 W Remove the appropriate number of microtife wells from the foil pouch and place them into the well
6. 0 pL 5 uL Total LOmL 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 sthe foi 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 partially purified recombinant human Mps1 add 10 uL of each fraction to the wells of the assay plate on ice Duplicate wells containing 20 m units 10 uL human Mps1 TTK positive control Cat Y E1I79 should be included in each assay as a positive control for phosphorylation 4 Begin the kifias yreaction by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer andifieubate at 30 C for 60 minutes 5 Wash wellssfave 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 Cat CY 1179 6 Version 140318 Human Mps 1 TTK kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual Te 8 9 For Research Use Only Not for use in diagnostic procedures and incubate at room temperature ca 25 C for 60 minutes Discard
7. 7 Mills GB Schmandt R McGill M Amendola A Hill M Jacobs K May C Rodricks AM Campbell S Hogg D Expression of Human Mps1 a novel human proteinekinasey is associated with cell proliferation J Biol Chem 267 22 16000 6 1992 8 Douville EM Afar DE Howell BW Letwin K Tannock L Ben Dawid Y Pawson T Bell JC Multiple cDNAs encoding the esk kinase predict transmembrane and intracellular enzyme isoforms Mol Cell Biol 12 2681 9 1992 9 Liu ST Chan GK Hittle JC Fujii G Lees E Yen TJ Humian MPS1 kinase is required for mitotic arrest induced by the loss of CENP E from kinetochores Wol Biol Cell 14 1638 51 2003 10 Olesen SH Thykjaer T Orntoft TF Mitotic checkpoint genes hBUB1 hBUB1B hBUB3 and Human Mps1 in human bladder cancer screening for mutations and loss of heterozygosity Carcinogenesis 22 5 813 5 2001 11 Hogg D Guidos C Bailey D Amendola A Groves Ty Davidson J Schmandt R Mills G Cell cycle dependent regulation of the protein kinase Human Mpst Oncogene 9 1 89 96 1994 12 Schmandt R Hill M Amendola A Mills GB Hogg D IL 2 induced expression of Human Mps1 a serine threonine tyrosine kinase correlateswith cell cycle progression J Immunol 152 1 96 105 1994 13 Iwase T Tanaka M Suzuki M Naito Y Sugimura H Kino I Identification of protein tyrosine kinase genes preferentially expressed in embryo stomach and gastric cancer Biochem Biophys Res Commun 194 2 698 705 1993 Jul 30 1993 14 Castillo
8. Positive Control 2 munit uL 10 uL 10 uL 10 uL or your enzyme fraction 10X Staurosporin See page 4 section Material Required but not Provided Cat CY E1179 See page 4 section Materials Required but not Provided 1 Following the above table add the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of Diluted human Mps1 TTK positive control to each well and mixing thoroughly at room temperature Coverywith plate sealer Incubate at 30 C for 60 minutes 2 Follow the Stahdard Assay steps 5 10 page 6 7 Cat CY 1179 8 Version 140318 Human Mps 1 TTK kinase Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures F 4 YCLEX Special considerations when measuring precise Human Mps1 activity In order to measure the activity of human Mps1 TTK 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 human Mps1 TTK enzyme activity is inthe sample the high level of A450 is not observed in Inhibitor control ATP minus contg l and No enzyme control Assay reavenis Test Inhibitor ATP minus Positive
9. abl from CycLex Cat CY E1179 One vial contains 4 units 200 uL human Mps1 enzyme Positive control should be added to the first well at 10 m units well For instance diluted positive control 1 10 use 10 uL for 1 assay Unused human Mps1 enzyme should be stored in aliquots at 70 C e 10X Staurosporine 10 uM Staurosporine 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 2004000 uE 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 meastifing absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 55Q 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 e Reagent reservoirs Deionized watenof thehighest quality e Disposable paper towels Cat CY 1179 4 Version 140318 Human Mps 1 TTK kinase Assay Inhibitor Screening Kit F 4 P 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 reagentsato excessive light Avoid freeze thaw cycles e Allow all the compone
10. actions on graph paper plot the meanjabsorbance 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 human Mps1 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 Research Product CycLex Human Mps1 TTK kinase Assay Inhibitor Screening Kit has been shown to detect the activity of recombinant human Mps1 in c6lumn fractions The assay shows good linearity of sample response The assay may be used to follow the purification of recombinant human Mpsl1 Troubleshooting 1 The human Mps1 TTK positive control should be piin duplicate 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 kinetic of the assay is of the first order Variations in the protocol can lead to non linearity of the elifve as can assay kinetics that are other than first order For a non linear curve point to point or quadratic urve 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 wash
11. d 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 1179 3 Version 140318 Human Mps 1 TTK 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 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 human Mps 1 substrate as a 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 12 mL of HRP horseradish peroxidase conjugated anti phospho serine monoclonal antibody TK 21B 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 Human Mps1 TTK Positive Control A vail
12. e as in step 6 9 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 10 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 wavelengths 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 human Mps1 TTK activity im 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 human Mps1 TTK 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 lt 0 2 A 7 t Test sampl Solvent Inhibitor ici TEABES Can Sap control control Kinase Reaction Buffer 80 uL 80 uL 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 uL 10X Staurosporin 10 uM 10 uL Human Mps1 TTK
13. ein kinase inhibitor Staurosporine on activity of recombinant human Mps1 oO v 120 0 p IC50 300 n M 100 0 80 0 60 0 Intensity of control 20 0 0 00 0 01 0 10 1 00 10 00 100 00 Staurosporine uM C CY 1179 12 Version 140318 Human Mps 1 TTK kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References j Stucke VM Sillje HH Arnaud L Nigg EA Human Mps kinase is required for the spindle assembly checkpoint but not for centrosome duplication EMBO J 21 7 1723 32 2002 2 Abrieu A Magnaghi Jaulin L Kahana JA Peter M Castro A Vigneron S Lorca T Cleveland DW Labbe JC Mps1 is a kinetochore associated kinase essential for the vertebrate mitoti checkpoint Cell 106 1 83 93 2001 3 Lauze E Stoelcker B Luca FC Weiss E Schutz AR Winey M Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase EMBO J 1995 Apr 18 14 8 1655 63 4 Winey M Goetsch L Baum P and Byers B MPS1 and MPS2 novel yeast gemes defining distinct steps of spindle pole body duplication J Cell Biol 114 745 754 1991 5 Weiss E Winey M The Saccharomyces cerevisiae spindle pole body duplication gene MPS is part of a mitotic checkpoint J Cell Biol 132 111 23 1996 6 Fisk HA Winey M The mouse Mps 1p like kinase regulates centrosome duplication Cell 106 95 104 2001
14. er maintenance 4 Overall low signal may indicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent D 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 Human Mps1 TTK kinase Assay Inhibitor Screening Kit have 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 must be stored at 20 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containingsa desiccant pack For researchwse only not for use in diagnostic or therapeutic procedures Cat CY 1179 10 Version 140318 wn Human Mps1 TTK kinase Assay Inhibitor Screening Kit Ce ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant human Mps1 enzyme reaction 3 5 7 0 20 40 60 80 Recombinant Mps1 TTK mU wy Fig 2 Time course of recombinant human Mps1 enzy action 18 p 0 0 1 1 1 1 1 1 0 20 40 60 80 100 120 Reaction time min C CY 1179 11 Version 140318 LA Human Mps1 TTK kinase Assay Inhibitor Screening Kit NyCLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of broad prot
15. in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substfate in the presence of Mg and ATP The amount of phosphorylated substrate is measured by binding tt with a horseradish peroxidase conjugate of TK 21B an anti phospho serine specific antibody which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a_coldrless solution to a blue solution or yellow after the addition of stopping reagent The eolor is quantified by spectrophotometry and reflects the relative amount of human Mps1 actiyity i t e sample For kinetic analysis the sample containing human Mps1 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 Human Mps1 TTK kinase Assay Inhibitor Screening Kit is designed to accurately determine the presence and relative amount of human Mps1 kinase activity in purification column fractions and to determine non isotopic kinetic analysis of human Mps1 kinase activity Careful attention to methods of chromatography and the assay protocol will provide the investigator with a reliable tool for the evaluation of human Mps activity Summary of Procedure Add 100 uL of sample to the wells 4 Incubate for 60 min at 30 C Wash the wells t Add 100 uW6fHRP conjugate
16. inhibitors or activatoxs of human Mps1 TTK 2 Detecting the effects of pharmacological agents on human Mps1 TTK 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 1179 1 Version 140318 Human Mps 1 TTK kinase Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Recently the vertebrate orthologues of the yeast MPS1 kinase were identified and found to localizesto kinetochores 1 2 MPS1 encodes a tyrosine and serine threonine dual specificity kinase 3 that was originally identified in a genetic screen for mutants defective in spindle pole duplication 4 Subsequently it was discovered to be an essential component of the mitotic checkpoint 5 Consistent with yeast MPS1 mouse Mps is localized at centrosomes throughout the cell cycle and is essential for accurate centrosome duplication 6 However a recent study indicated that human Mps was not localized at centrosomes in human U2OS cells 1 Despite the discrepancy in the centrosome localization of Mps1 in mouse and human cells it is clear that Mps1 is present at kinetochores during mitosis where it may participate in the checkpoint The human Mps1 TTK kinase was originally identified in a screen for novel tyrosine kifiases b
17. nts to come to room temperature before use e 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 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 e The buffers and reagents in this kit may contain preservatives of other chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipet or ingest any of the reagents e Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containing 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 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 e CAUTION Sulfuric_Acidyis a strong acid Wear disposable gloves and eye protection when handling Stop
18. p Human Mps1 TTK kinase Assay Inhibitor Screening Kit w kd c ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring Human Mps1 TTK Activity CycLex Human Mps1 TTK kinase Assay Inhibitor Screening Kit Cat CY 1179 Mmtended Use sccescieeetececteenisiiee eee 1 SOS yiee eee eE E EES 1 Tntroductio feen nnn e 2 Principle of the Assay 3 Materials Provided cceccceeeeeeeeeeeeseees 4 Materials Required but not Provided 4 Precautions and Recommendation 5 Detailed Protocol ecceeccsceeseeeeeeeeeees 6 9 Evaluation of Results cccccceeeeeeeeeeeee 10 Assay Characteristics 10 Troubleshooting seeseeeeeeeeeeereerrerrerrrrreeee 10 Reagent Stability 10 Example of Test Resilts csicssssssssslesevoectenaces 14 12 References iscdeistisicssisveadl cist tian Senveks 13 Related Products ccccceeececeeesseseeeeeees 14 Intended Use The CycLex Research Product CycLex Human Mps1 TTK kinase Assay Inhibitor Screening Kit is designed to measure the activities of putified human Mps1 TTK 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 thegphospho serine residue in recombinant human Mps 1 substrate which is phosphorylated by human Mps METK Applications of this kit include 1 Screening
19. sitive 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 onan infrequent basis The CycLex Research Product CycLex Human Mpsi TTK kinase Assay Inhibitor Screening Kit uses a peroxidase coupled anti phospho serine specific monoclonal antibody as a reporter molecule in a 96 well ELISA format This assay provides a non isotopic sensitive and specific method to detect human Mps1 TTK activity Cat CY 1179 2 Version 140318 Human Mps 1 TTK 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 Human Mps1 TTK kinase Assay Inhibitor Screening d it is a single site semi quantitative immunoassay for human Mps1 TTK activity Plates are pre coated with a substrate corresponding to recombinant human Mpsl substrate newly designed by_CycLex which contains a serine residue that is phosphorylated by human Mps1 TTK The detector antibody specifically detects only the phosphorylated form of serine residue on human Mps1 TTK substrate The CycLex Research Product CycLex Human Mps1 TEK Kinase Assay Inhibitor Screening Kit can be used to study the kinetics of a purified or partially purified human Mps as well as to screening these kinases inhibitor To perform the test the sample is diluted
20. y using a phosphotyrosine antibody to screen a T cell cDNA expression library 7 Usingya similar strategy the mouse homologue esk was also cloned from an embryonal carcinoma cell line 8 Mt was determined subsequently that esk was the mouse orthologue of yeast MPS1 6 Human Mps1 is detectable in all proliferating human cells and tissues Expression of the human Mps1 geneds markedly reduced or absent in resting cells and in tissues with a low proliferative index Levels of human Mps1 mRNA and protein are very low in starved cells When cells are induced to enter the cell cyel levels of human Mps1 mRNA protein and kinase activity increase at the G1 S phase of the cell cycle and peak in G2 M Human Mps1 mRNA levels as well as kinase activity drop sharply in early G1 whereas protein levels are largely maintained Human Mps1 may play a role in cell cycle control Measurement of Human Mps1 TTK activity The protocol generally regarded as most sensitive for the quantitative measurement of human Mps1 TTK activity involves incubation of the hunian Mps1 TTK sample with substrate either a natural or synthetic polypeptide such as MBP in the presence of Mg and P labeled ATP The reaction is terminated by spotting a sample onto a filter paper dis 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 sen

CY-1179

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