Data Sheet
Contents
1. 1204202. 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 Note 1 Complete removal of liquid at each stepaisyessential to good performance 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 3 units for the blank no enzyme control or 2 5 units for the 50 m units well of Plk2 positive control Cat CY E1183 Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of the Plk2 positive control perform a second reading at 405 nm A new O D values measured at 405 nm is used to d t rmine P1k2 activity of off scale samples The readings at 405 nm should not replace the on s ale readings at 450 nm Kinetic Assays 1 Remove theappropriateynumber of microtiter wells from the foil pouch and place them into the well holder Returmany unused wells to the foil pouch refold seal with tape and store at 4 C 2 Prepare allsamples diluted with Kinase Buffer as needed 3 Add 10 pL of individual purified Plk2 sample to the well of the assay plate on ice Duplicate wells containing 50 m units 10 uL of Plk2 positive control Cat CY E1183 should be included in each assay aSja positive control for phosphorylation 4 Begin the kin
3. Buffer provided to 900 mL of ddH 0 Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH O to the vial of 20X ATP provided lyophilized Mix gently until dissolved The final concentration ofthe 20X ATP Solution should be 1 25 mM Store the solution in small aliquots e g 100 uL at 20 C 3 Prepare Kinase Reaction Buffer DTT amp ATP plus by mixing following reagents 96 assays 10 assays 1 assay Kinase Buffer provided 9 0 mL 900 uL 90 pL 20X DTT Solution provided 0 5 mL 50 uL 5 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 DTT amp ATP plus per assay well Mix well Discard any unused Kinase Reaction Buffer DTT amp ATP plus 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 All samples should be assayed in duplicate 3 Add 10 pimof individual purified PIk2 sample to the well of the assay plate on ice Duplicate wells containing 50 m units 10 uL of Plk2 positive control Cat CY E1183 should be included in each asSay as a positive control for phosphorylation 4 Begin theykinase reaction by addition of 90 uL K
4. Hi yclex PIk2 positive control 5 m units uL or Purified enzyme sample For Research Use Only Not for use in diagnostic procedures 10 uL 10 uL 10 uL Calbiochem Cat 528282 See Page 4 section Materials Required but not Provided Plk2 positive control Cat CY E1183 See Page 5 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 Plk2 positive control 5 m units pL to each well and mixing thoroughly at room temperature Cover with plate sealer or lid and incubate at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 12 page 7 8 Note Although we suggest to conduct experiments as outlined in the table above the optimal experimental conditions will vary depending on the parameters being Investigated and must be determined by the individual user Especially appropriate amount of Plk2 positive control must be determined by titration of the Plk2 positive control and setting the amount which shows OD value does not exceed plateau range in dose response curve NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND MUST gt BE DETERMINED BY THE INDIVIDUAL USER NO WARRANTY lt OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED Special co
5. RP conjugated Anti rabbit IgG One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti rabbit IgG 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 NHgSO Ready to use Cat CY 1183 4 Version 120420 pry Polo like kinase 2 Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e PIk2 Positive Control Available from CycLex Plk2 Positive Control Cat CY E1183 One vial containing 10 units 100 uL 100 m units uL Plk2 enzyme Positive control should be added to the first well at ca 50 m units well Unused Plk2 enzyme should be stored in aliquots at below 70 C 10X PIk Inhibitor I 250 uM Plk Inhibitor I is available from Calbiochem Cat 528282 10 mM stock solution DMSO diluted 1 40 in Kinase Buffer e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Wash bottle or multichannel dispenser for plate washing e Microcentrifuge and tubes for sample preparation e Vortex mixer e Plate reader capable of measuring absorbance in 96 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 wh
6. ase reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervals suggested interval is 4 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 Cat CY 1183 8 Version 120420 pry Polo like kinase 2 Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 5 Stop the reaction by flicking out the contents Alternatively the reaction may be terminated bysthe addition of 150 uL 0 2 M Na EDTA pH 8 0 to each 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 Anti Phospho Threonine Polyclonal Antibody PPT 08 into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 60 minutes Discard any unused antibody after use o0 Wash wells five times as same as in step 6 9 Pipette 100 uL of HRP conjugated Anti rabbit IgG into each well coverwith plate sealer or lid and incubate at room temperature ca 25 C for 60 minutes Discardjany unused conjugate after use 10 Wash wells five times as same as in step 8 11 Add 100 uL of Substrate Reagent to each well and incubate abroom temperature ca 25 C for 5 15 minutes Appropriate incubation time may vary and incubation time can be ext
7. ce 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 when necessary 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 Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1183 6 Version 120420 yy YCLEX Detailed Protocol User s Manual Polo like kinase 2 Assay Inhibitor Screening Kit For Research Use Only Not for use in diagnostic procedures The CycLex Polo like kinase 2 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 Since conditions may vary running an aliquot of the appropriate PIk2 positive control Cat CY E1183 separately available from CycLex 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
8. e is nearly a straight line if the kinetics othe 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 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 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 Polo like kinase 2 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 containing a desiccant pack Forsresearch use only not for use in diagnostic or therapeutic procedures Cat CY 1183 11 Version 120420 pry Polo like kinase 2 Assay Inhibitor Screening Kit H y 3X User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Result
9. ended up to 20 minutes if the reaction temperature is below than 20 C 12 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 13 Measure absorbance in each well using a spectorphotometric plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Recommendations Special considerations when screening activators or inhibitors In order to estimate the inhibitory effect on individual Plk2 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 Whenetest chemicals cause an inhibitory effect on Plk2 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 5 Assay reagents Test sample Bew pawei Kinase Reaction buffer DTT amp ATP plus 80 uL 80 uL 80 uL 10X Inhibitor or equivalent 10 pL Vehicle for Inhibitor 10 pL 10X Plk Inhibitor I 250 uM 10 uL Cat CY 1183 9 Version Polo like kinase 2 Assay Inhibitor Screening Kit User s Manual
10. ening Kit is a single site semi quantitative immunoassay for Plk2 activity Plates are pre coated with a substrate corresponding to recombinant Plk2 substrate which contains threonine residues that can be efficiently phosphorylated by Plk2 The detector antibody specifically detects only the phosphorylated form of threonine residue on Plk2 substrate The CycLex Polo like kinase 2 Assay Inhibitor Screening Kit may be used to study the kinetics of a purified PIk2 as well as to screening Plk2 inhibitor or activator To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substrate following the addition of Mg and ATP The amount of phosphorylated substrate is measured by binding it with a PPT 08 a anti phospho threonine polyclonal antibody followed by binding with horseradish peroxidase conjugated anti rabbit IgG 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 Thejcolor is quantitated by spectrophotometry and reflects the relative amount of Plk2 activity in the sample For kinetic analysis the Plk2 containing sample is added to the wells in a similar fashion and at varying times the reaction is stopped by the addition of the chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as befo
11. gan I amp Glover D M Genes Dev 9 1059 1073 1995 5 Song S amp Lee K S J Cell Biol 152 451 469 2001 6 Simmons DL Neel BG Stevens R Evett G et al Mol Cell Biol 12 4164 4169 1992 7 Kauselmann G M Weiler P Wulff et al EMBO J 18 5528 5539 1999 8 Ma S M Y Liu Y L O Yuan and R L Erikson Mol Cancer Res 1 376 384 2003 9 Ma S Charron J Erikson R Mol Cell Biol 23 6936 6943 2003 10 S Warnke S Kemmler RS Hames HL Tsai et al Curr Biol 1431200 1207 2004 11 Burns TF Fei P Scata KA et al Mol Cell Biol 23 5556 5571 2003 Related Products Plk1 Positive control Cat CY E1163 Plk2 Positive control Cat CY E1183 Plk3 Positive control Cat CY E1176 CycLex Polo like kinase 1 Assay Inhibitor Screening Kit Cat CY 1163 CycLex Polo like kinase 2 Assay Inhibitor Screening Kit Cat CY 1183 CycLex Polo like kinase 3 Assay Inhibitor Screening Kit Cat CY 1176 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 whittp www cyclex co 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 1183 14 Version
12. ich will give a somewhat higher reading 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels Cat CY 1183 5 Version 120420 Polo like kinase 2 Assay Inhibitor Screening Kit yy ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Store the Plk2 enzyme at below 70 C and the ATP at 20 C when not in use Store all other components at 4 C Do not expose reagents to excessive light Avoid freeze thaw cycles 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 e The buffers and reagents used in this kit contain either sodium Kathon CG as preservatives Care should be taken to avoid direct contact with these reagents Do not mouth pipette 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 complian
13. inase Reaction Buffer per well cover with plate sealer or lid and incubate at 30 C for 60 minutes Nn residual Wash Buffer by gentle tapping or aspiration Cat CY 1183 T Version Wash wells five times with Wash Buffer making sure each well is filled completely Remove 120420 P Polo like kinase 2 Assay Inhibitor Screening Kit oy GA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 6 Pipette 100 uL of Anti Phospho Threonine Polyclonal Antibody PPT 08 into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 60 minutes Discard any unused antibody after use 7 Wash wells five times as same as in step 5 8 Pipette 100 uL of HRP conjugated Anti rabbit IgG 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 use 9 Wash wells five times as same as in step 7 10 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 5 15 minutes Appropriate incubation time may vary and incubation time can be extended up to 20 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each well using a spectrophotomettic plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or
14. it has been demonstrated to recognize the phospho threonine residue in recombinant Plk2 substrate which is efficiently phosphorylated by P1k2 Plk2 substrate is CycLex s intellectual property Applications ofthis kitinclude 1 Screening inhibitors Or activators of Plk2 2 Detecting the effects of pharmacological agents on P1k2 activity This assay kitis 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 1183 1 Version 120420 Polo like kinase 2 Assay Inhibitor Screening Kit wy cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Polo like kinases Plks have been shown to be important contributors to several cell cycle ev nts 2 Genetic and biochemical experiments in various organisms indicate that polo like kinases regulate diverse cellular events at multiple mitotic stages Genetic studies in Drosophila and yeast indicate plks function in centrosome assembly and separation during the formation of the bipolar spindle Drosophila polo mutants reveal phenotypes of hyper condensed chromosomes monopolar spindles disorganized spindle poles and abnormal chromosome segregation Schizosaccharomyces pombe plol displays similar phenotypes such as the formation of monopolar spindles or a failure in septum formation after n
15. nsiderations when measuring precise Polo like kinase 2 activity In order to measure the activity of Plk2 correctlysit 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 PIk2 enzyme activity is in the sample the high level of A450 is not observed in Inhibitor control ATP minusycontrol and No enzyme control Assay reagents Test Inhibitor ATP minus Positive No enzyme Sample control control control control Kinase Reaction buffer DTT amp ATP plus 80 uL 80 uL 80 uL 80 uL Kinase Buffer DTT plus ATP minus 80 uL 10X PIk Inhibitor I 250 uM 10 pL Vehicle for 10X Compound 10 uL 10 uL 10 uL 10 uL Your enzyme sample 10 uL 10 uL 10 uL PIk2 positive control 6m units uL 10 pL Buffer for your enzyme sample 10 pL Calbiochemy Cat 528282 See Page 4 section Materials Required but not Provided Plk positive control Cat CY E1183 See Page 5 section Materials Required but not Provided 1 4Following the above table add the Reagents to each well of the microplate Finally initiate the reaction by adding 10 uL of Your enzyme sample or Buffer to each well and mixing
16. pry Polo like kinase 2 Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring Plk2 Activity CycLex Polo like kinase 2 Assay Inhibitor Screening Kit Cat CY 1183 Intended WS lt scsctecssorvocessanssaeensveceanrvessesenenss 1 PI MOEAL Gs suistscuusy bccn sasasunecsesapnedauntucionodesentevaonines 1 WTEC TOM 2554 ccanhdesdccaciecaceapabarnctalcenieasaage 2 Principle of the Assay ccccsscesseeseeeees 3 Materials Provided 4 sissscciscsavieatisavidesstaversncs 4 Materials Required but not Provided 5 Precautions and Recommendation 6 Detailed Protocol vssssssicdcnissiaiaressseessieeisaiane 7 10 Evaluation of Results cccccccecessseeees 11 Assay Characteristics saciesnntsasuntoncvumseventonae 11 Troubleshooting veces vcsscnaadesbeacaeeccasiecnts 11 Reagent Stability ccc cecceceseeseesteeees 11 Example Of Test Result sscanitosuseennssornienaay 12 13 References cesseesseseeseseeeesseeeessreeesssreesesseee ME 14 Related Products cccccscccceesseeeeceeetiny es 14 Intended Use The CycLex Research Product Cyebex Polo like kinase 2 Assay Inhibitor Screening Kit is designed to measure the activities of purified Polo like kinase 2 PIk2 for the rapid and sensitive evaluation of inhibitors using recombinant Plk2 The phospho threonine specific polyclonal antibody used in this assay k
17. re Summary of Procedure Add 100 uL of sample to the wells Vv Incubate for 60min at 30 C Wash the wells Add 100 uL of antiephospho threonine polyclonal antibody PPT 08 Vv Incubate for 60 min at room temp Wash th wells Add 100 uL of HRP conjugated anti rabbit IgG y Incubate for 60 min at room temp Wash the wells v Add 100 uL of Substrate Reagent v Add 100 pL of Stop Solution v Measure absorbance at 450 nm Cat CY 1183 3 Version 120420 Polo like kinase 2 Assay Inhibitor Screening Kit yy 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 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 a foil zip lock bag with a desiccant pack Wells are coated with recombinant Plk2 substrate as Plk2 substrate 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Kinase Buffer One bottle containing 20 mL of Kinase Buffer Used for Kinase Reaction Buffer and sample dilution 20X DTT Solution One tube containing 1 2 mL of 20X DTT Solution 20X ATP Lyophilized ATP Na salt Anti Phospho Threonine Polyclonal Antibody PPT 08 One bottle containing 12 mL of anti phospho threonine polyclonal antibody PPT 08 Ready to use H
18. s Fig 1 Dose dependency of recombinant Plk2 enzyme reaction PIk2 dose dependency 3 0 A450 0 25 50 75 100 PIk2 dose mUnit Fig 2 Dose dependency of ATP Dose dependency of ATP 3 0 2 5 2 0 N 1 5 1 0 0 5 0 0 0 20 40 60 80 100 ATP conc uM Cat CY 1183 12 Version 120420 7 yclex Polo like kinase 2 Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Km for ATP recombinant P1k2 A 2 Nn V om N lt f lt Km for ATP 0 y 10 467x 79 938 R 0 9854 Km for ATP 7 6 uM 10 20 30 40 50 60 70 80 90 100 ATP conc uM Fig 4 Effect of Polo like Kinase specific inhibitorsPolo like Kinase Inhibitor I Calbiochem on P1k2 activity Inhibition by Plk Inhibitor I 2 0 9 1 5 Z t lt 10 0 5 0 0 0 5 10 15 20 25 PIk Inhibitor I conc uM Cat CY 1183 13 Version 120420 pry Polo like kinase 2 Assay Inhibitor Screening Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References pa Lane H A amp Nigg E A Trends Cell Biol 7 63 68 1997 2 Glover D M Hagan I M amp Tavares A A M Genes Dev 12 3777 3787 1998 3 Sunkel C E amp Glover D M J Cell Sci 89 25 38 1988 4 Ohkura H Ha
19. thoroughly at room temperature Cover with plate sealer or lid and incubate at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 12 page 7 8 Cat CY 1183 10 Version 120420 pry Polo like kinase 2 Assay Inhibitor Screening Kit os ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Average the absorbance values for the PIk2 sample duplicates positive control and all experimental sample duplicate values when applicable When P1k2 positive control 50 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 30 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 Polo like kinase 2 Assay Inhibitor Screening Kit has been shown to detect the activity of purified recombinant Plk2 The assay shows good linearity of sample response Troubleshooting The CycLex PIk2 positive control Cat CY E1183 should be run i 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 curv
20. tly induced by wild type p53 after DNA damage and in these circumstances activates a G2 checkpoint Measurement of Polo like kinase 2 activity The protocol generally regarded as most sensitive for the quantitative measurement of Plk2 activity involves incubation of the Plk2 sample with exogenous substrate such as a casein or MBP in the presence of Mg and P labeled ADP Thesreaction 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 a radioisotope of short half life It is particularly unsuitable when kinase assays are only performed on an infrequent basis The CycLex Polo like kinase 2 Assay Inhibitor Screening Kit uses anti phospho threonine polyclonal antibody PPT 08 and peroxidase coupled anti rabbit IgG antibody as a reporter molecule in a 96 well ELISA format This assay provides a non isotopic sensitive and specific method to measure the activities of PIk2 Cat CY 1183 2 Version 120420 P Polo like kinase 2 Assay Inhibitor Screening Kit oy cdl ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex Polo like kinase 2 Assay Inhibitor Scre
21. uclear division The budding yeast poleclike kinase homolog Cdc5 seems to play anamportant role in actin ring formation and cytokinesis Plk2 also called Snk a member of polo like kinase family was originally identified as immediate to early transcripts in mouse fibroblasts however it is expressed at mainly in the hippocampal neurons in the brains of adult rodents The level of Plk2 in the brainsis increased by stimuli that induce long term potentiation When ectopically expressed Plk2 caused changes in cell morphology and after an extended period of time cell death The precise function of Plk2 is uncertain However from analysis of Plk2 mice it appears that the protein has a role in development and is involved in cell cycle progression Plk2 protein is expressed at equal abundance during G1 S and G2 phases in HeLa cell In contrast Plk2 kinase activity peaked as near the Gl to S transition and decreased again as cells efitered G2 phase of the cell cycle Endogenous P1k2 localize with centrosomes and this interaction is independent of Plk2 kinase activity In contrast the kinase activity of Plk2 is required for centriole duplication These results show that PIk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1 to S phase transition There is also evidence that Plk2 functions as a stress response protein Expression of Plk2 is direc
Download Pdf Manuals
Related Search