CycLex Polo-like kinase 2 Assay/Inhibitor Screening Kit
Contents
1. Wash wells five times as same as in step 6 y oo Wash wells five times as same as in step 6 11 Add 100 uL of Substrate Reagent to each w d incubate at room temperature ca 25 C for 5 15 minutes Appropriate incubation time vary and incubation time can be extended up to 20 minutes if the reaction temperature is belgoj than 20 C Ww 12 Add 100 uL of Stop Solution to apel in the same order as the previously added Substrate Reagent KX 13 Measure absorbance in each well asing a spectorphotometric plate reader at dual wavelengths of 450 540 nm Dual wavelengths 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wavelen an be used Wells must be read within 30 minutes of adding the Stop Solution a Roa CY 1183 9 Version 150629 o c yclex Polo like kinase 2 Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures O Special considerations when screening activators or inhibitors In order to estimate the inhibitory effect on individual Plk2 activity in the test chemicals c tly it is necessary to conduct the control experiment of Solvent control at least once for every e iment and Inhibitor control at least once for the first experiment in addition to Test sample as ggdicated in the Recommendations ww Q O Q gt following table When test chemicals cause an inhibitory effect on Plk2 activi2. 0 5 10 15 20 25 Plk Inhibitor I conc aM gL A Roa CY 1183 14 Version 150629 o c g Polo like kinase 2 Assay Inhibitor Screening Kit f cle User s Manual v 3X y For Research Use Only Not for use in diagnostic procedures F References 1 Lane H A amp Nigg E A Trends Cell Biol 7 63 68 1997 we 2 Glover D M Hagan I M amp Tavares A A M Genes Dev 12 3777 3787 1998 RY 3 Sunkel C E amp Glover D M J Cell Sci 89 25 38 1988 g 4 Ohkura H Hagan I amp Glover D M Genes Dev 9 1059 1073 1995 2 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 4166o 1992 Kauselmann G M Weiler P Wulff et al EMBO J 18 5528 5530 09 S 8 Ma S M Y Liu Y L O Yuan and R L Erikson Mol Cancef es 1 376 384 2003 9 Ma S Charron J Erikson R Mol Cell Biol 23 6936 6943 W 10 S Warnke S Kemmler RS Hames HL Tsai et al Curr Bil 14 1200 1207 2004 11 Burns TF Fei P Scata KA et al Mol Cell Biol 23 6956 5571 2003 Related Products Q PIk1 Positive control Cat CY E1163 PIk2 Positive control Cat CY E1183 amp PIk3 Positive control Cat CY E117 CycLex Polo like kinase 1 Assay Inifbitor Screening Kit Cat CY 1163 CycLex Polo like kinase 2 Assay I hibitor Screening Kit Cat CY 1183 CycLex Polo like kinase 3 Assqfihibitor Scre
3. 4 Incub 60 min at 30 C Wash the wells NW Add 100 uL of ote oni polyclonal antibody PPT 08 amp x Waspghe wells o Add Au of HRP conjugated anti rabbit IgG Q J Incubate for 60 min at room temp Wash the wells S N Add 100 uL of Substrate Reagent Oe 4 O Add 100 pL of Stop Solution Y Q Measure absorbance at 450 nm amp gL G Roa CY 1183 3 Version 150629 S C ncubate for 60 min at room temp Polo like kinase 2 Assay Inhibitor Screening Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Materials Provided 3 All samples and standards should be assayed in duplicate The following components are uta and are sufficient for the one 96 well microtiter plate kit RY Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in yfoil zip lock bag with a desiccant pack Wells are coated with recombinant Plk2 substrate as PIk2 trate 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 wrez Kinase Buffer One bottle containing 20 mL of Kinase Buffer Used for Kiffa e Reaction Buffer and sample dilution Y 20X ATP One vial of lyophilized ATP Naz salt Ro 20X DTT Two vials of lyophilized dithiothreitol N Anti Phospho Threonine Polyclonal Antibody One Vile containing 12 mL of anti phospho threonine polyclonal antibody PPT 08 Ready Quse HRP conjugated Anti rabbit IgG On
4. S Polo like kinase 2 Assay Inhibitor Screening Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Non Radioisotopic Kit for Measuring PIk2 Activity l CycLex Polo like kinase 2 Assay Inhib r Screening Kit Cat CY 1183 2 gt Intended USse ccceceeccccccececesseseseeeesseeeeees 1 ne Be rat tabsincasega yonenysanagocsiciasiadsoaemeiaaeeansinen 1 a Introduction sssssessseeeesesesereresrersrserrsrreresreses 2 Yon Principle Of the Assay 3 gt Materials Provided cceccceeeeseeeeeeeeeees 4 Materials Required but not Provided 5 N Precautions and Recommendations 6 4 Detailed Protocol ccccccccccececeecssssseeeeeeeees 7 10 2 Evaluation of Results w c cc eeeeeeeeeeeees 11 4 Assay Characteristics sses 11 gt Troubleshooting sscusnossstsncesonsoanensnanbncanniics 11 Reagent Stability ssccssssonesavanatvncsnrramnsonannouess we Example of Test Result eee eeeeeeeees WG 13 RefErenCES s cc2cczsssssssechccceevesessisssseacetcanceengee 4 Related Products ccccceeeeeeeeesceeeseeene o 14 Intended Use amp The CycLex Research Product Lex Polo like kinase 2 Assay Inhibitor Screening Kit is designed to measure the activities purified Polo like kinase 2 PIk2 for the rapid and sensitive evaluation of inhibitors using red pmbinant Plk2 The phospho threonine specific polyclonal antibody used in this assay kit has beens o
5. Y DEPENDING ON THE PARAMETERS EING INVESTIGATED AND MUST BE DETERMINED BY THE Phi DUAD USER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING T E PROCEDURES IS MADE OR IMPLIED F amp a NOTE THE IAL EXRARIMENTAL ARE INTENDED ONLY AS A GUIDELINE THE A Roa CY 1183 10 Version 150629 o c ft YCLEX A Polo like kinase 2 Assay Inhibitor Screening Kit User s Manual A For Research Use Only Not for use in diagnostic procedures Special considerations when measuring precise Polo like kinase 2 activity In order to measure the activity of PIk2 correctly it is necessary to conduct the control experimen of Inhibitor control at least once for every experiment and ATP minus control at least once first experiment in addition to No enzyme control as indicated in the following table Although ta level of A450 increases in Test sample when Plk2 enzyme activity is in the sample the high lev 6f A450 is not observed in Inhibitor control ATP minus control and No enzyme control amp Assay reagents Test Inhibitor ATP minus Posttive No enzyme Sample control control rol control Kinase Reaction buffer 80 uL 80 pL oe pL 80 uL Kinase Buffer provided 80 Bh 10X Pik Inhibitor I 250 uM 10 pL a Vehicle for 10X Compound C 10 uL 10 uL 10 uL Your enzyme sample 10 uL 10 uL uL PIk2 positive control 5 m units uL QY 10 uL Buffer f
6. ast polo like kinase homolog Cdc5 seems ta play an important role in actin ring formation and cytokinesis Yon PIk2 also called Snk a member of polo like kinase family riginally identified as immediate to early transcripts in mouse fibroblasts however it ig eXPressed at mainly in the hippocampal neurons in the brains of adult rodents The level of PIk2 in N brain is increased by stimuli that induce long term potentiation When ectopically expressed gk caused changes in cell morphology and after an extended period of time cell death amp The precise function of Plk2 is uncertain However from analy f Plk2 mice it appears that the protein has a role in development and is involved in cell cycle ession PIk2 protein is expressed at equal abundance during G1 S and G2 phases in HeLa cell gn Contrast Plk2 kinase activity peaked as near the Gl to S transition and decreased again as cells pitered G2 phase of the cell cycle Endogenous Plk2 localize with centrosomes and this inter4 tion is independent of Plk2 kinase activity In contrast the kinase activity of Plk2 is required for cerable duplication These results show that PIk2 is a physiological centrosomal protein and that its kingye activity is likely to be required for centriole duplication near the G1 to S phase transition is also evidence that Plk2 functions as a stress response protein Expression of Plk2 is directly iNteced by wild type p53 after DNA damage and
7. been shown to detect the activity of purified recombinant PIk2 The assay ws good linearity of sample response amp a Troubleshooting 1 The CycLex Plk2 positive control Cat CY E1183 should 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 specifi ay give erroneous results 2 The reaction curve is nearly a straight line if the ka tics of the assay is of the first order Variations in the protocol can lead to non linearity of the curvas can assay kinetics of other than first order For a non linear curve point to point or quadratic e fit methods should be used 3 Poor duplicates accompanied by elevat ues for wells containing no sample indicate insufficient washing If all instructions in the Det Protocol were followed accurately such results indicate a need for washer maintenance lt 4 Overall low signal may indicate wpfftisication of the plate has occurred between the final wash and addition of Substrate Reagent Pa ot allow the plate to dry out Add Substrate Reagent immediately after wash amp a OA yoyo Reagent Stabilit All of the O i in the CycLex Research Product CycLex Polo like kinase 2 Assay Inhibitor Scfgching 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 excep
8. e bottle oito 12 mL of HRP horseradish peroxidase conjugated anti rabbit IgG Ready to use Substrate Reagent One bottle containing 20 m wf the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL roy H2SOs Ready to use v amp Roa CY 1183 4 Version 150629 o c A S Polo like kinase 2 Assay Inhibitor Screening Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Materials Required but not Provided 3 e PIk2 Positive Control Available from CycLex Plk2 Positive Control Cat CY E1183 S vial containing 10 units 100 uL 100 m units uL PIk2 enzyme Positive control should be d to the first well at ca 50 m units well Unused Plk2 enzyme should be stored in aliquots at bel 0 C 10X PIk Inhibitor I 250 uM Plk Inhibitor I is available from Calbiochem ca fan 10 mM stock solution DMSO diluted 1 40 in Kinase Buffer Y e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with ia tips Precision repeating pipettor aS gt 2 Y Vortex mixer N Plate reader capable of measuring absorbance in 96 wellghites at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can Aigo be used The plate can also be read at a single wavelength of 450 nm which will give a somewht higher reading 500 or 1000 mL graduated cylinder aS e Wash bottle or mult
9. ening Kit Cat CY 1176 ge PRODUCED BY CycLex Co Ltd S 1063 103 Terasawapig Ina Nagano 396 0 Japan Fax 181 265 7698 e mail info yzlex co j URL http w cyclex co j CyeLeg eux 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 pro s without prior written approval from CycLex Co Ltd To inquire about licensing for s ommercial use please contact us via email A Roa CY 1183 15 Version 150629 o c
10. ichannel dispenser for plate washing e Microcentrifuge and tubes for sample preparation e Reagent reservoirs xO Deionized water of the highest quality KA e Disposable paper towels amp Roa CY 1183 5 Version 150629 o c A S Polo like kinase 2 Assay Inhibitor Screening Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Precautions and Recommendations 3 e Store the PIk2 enzyme at below 70 C and the ATP at 20 C when not in use Storage other components at 4 C Do not expose reagents to excessive light Avoid freeze thaw cycles RY e Allow all the components to come to room temperature before use amp e All microplate strips that are not immediately required should be returned to the singe pouch which must be carefully resealed to avoid moisture absorption Q e Do not use kit components beyond the indicated kit expiration date y e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware RG e Use deionized water of the highest quality o e Do not mix reagents from different kits Ss e The buffers and reagents in this kit may contain preserv Bes or other chemicals Care should be taken to avoid direct contact with these reagents KS e Do not mouth pipette or ingest any of the reagents QY e Do not smoke eat or drink when performigg he assay or in areas where samples or reagents are handled re e Dis
11. in these circumstances activates a G2 checkpoint 1 Measurement of Polo like kinase 2 O The protocol generally regarded as sensitive for the quantitative measurement of Plk2 activity involves incubation of the Plk2 s e with exogenous substrate such as a casein or MBP in the presence of Mg and P labeled ne The reaction is terminated by spotting a sample onto a filter paper disc followed by immersiofQyn acid to precipitate the radiolabeled product The filter papers are then washed extensively to Gre unincorporated radiolabel and the radioactivity counted While sensitive this method is rer ntensive generates hazardous radioactive waste and depends on a radioisotope of short half life Yt is particularly unsuitable when kinase assays are only performed on an infrequent basis The cLex Polo like kinase 2 Assay Inhibitor Screening Kit uses anti phospho threonine Q lonal antibody PPT 08 and peroxidase coupled anti rabbit IgG antibody as a reporter molec a 96 well ELISA format This assay provides a non isotopic sensitive and specific method to ure the activities of Plk2 awe gt o G oe Roa CY 1183 2 Version 150629 o AO A S Polo like kinase 2 Assay Inhibitor Screening Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Principle of the Assay 3 The CycLex Research Product CycLex Polo like kinase 2 Assay Inhibitor Screening R is a single site
12. nstrated to recognize the phospho threonine residue in recombinant Plk2 substrate which is etQichtly phosphorylated by Plk2 A Plk2 substrate is Cagle intellectual property Applications of this clude 1 Screening inhibit fs or activators of Plk2 2 Detecting tigate of pharmacological agents on PIk2 activity for research use only and not for use in diagnostic or therapeutic procedures Storage e Up6s receipt store all components at 4 C 7 ign t expose reagents to excessive light A Roa CY 1183 1 Version 150629 o c Polo like kinase 2 Assay Inhibitor Screening Kit P 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction Polo like kinases Plks have been shown to be important contributors to several cell cycle Mis 12 Genetic and biochemical experiments in various organisms indicate that polo like S diverse cellular events at multiple mitotic stages Genetic studies in Drosophila and yeastemdicate plks function in centrosome assembly and separation during the formation of the bipolar sping Drosophila polo mutants reveal phenotypes of hyper condensed chromosomes monopolar spindl s disorganized spindle poles and abnormal chromosome segregation Schizosaccharomyces p e plol displays similar phenotypes such as the formation of monopolar spindles or a failure in s pjttm formation after nuclear division The budding ye
13. of individual purified Plk2 sample to the well of the assay plate on ice Duplicate wells co taining 50 m units 10 uL of Plk2 positive control Cat CY E1183 should be included in each ay as a positive control for phosphorylation e Reon CY 1183 8 Version 150629 S C A 4 Polo like kinase 2 Assay Inhibitor Screening Kit eA ycLex User s Manual A For Research Use Only Not for use in diagnostic procedures gt 4 Begin the kinase reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per welDin timed intervals suggested interval is 4 minutes but should be individually determined f ch system After the final addition cover with plate sealer or lid and incubate at 30 Kor 20 minutes N 5 Stop the reaction by flicking out the contents Alternatively the reaction may be geet by the 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 pletely Remove residual Wash Buffer by gentle tapping or aspiration va 7 Pipette 100 uL of Anti Phospho Threonine Polyclonal Antibody magi well cover with plate sealer or lid and incubate at room temperature ca 25 C for 60 nfwutes Discard any unused antibody after use Y 9 Pipette 100 uL of HRP conjugated Anti rabbit IgG into lt well cover with plate sealer or lid S and incubate at room temperature ca 25 C for 60 mip Discard any unused conjugate after use e2 10
14. or your enzyme sample 10 uL Calbiochem Cat 528282 See Page 4 section Materials Required bu Qs Plk2 positive control Cat CY E1183 See Page 5 section Materiaj Required but not Provided 1 Following the above table add the Reagents to ea ell of the microplate Finally initiate the reaction by adding 10 uL of Your enzyme sample gg Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer or lid alfa at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 12 pase O Roa CY 1183 11 Version 150629 o c Roa CY 1183 12 Version 150629 ge A S Polo like kinase 2 Assay Inhibitor Screening Kit Pe cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Evaluation of Results 3 1 Average the absorbance values for the PIk2 sample duplicates positive control and all ex Rhental sample duplicate values when applicable When Plk2 positive control 50 m units eel amie as an internal control for the phosphorylation reaction the absorbance value should be greage r than 1 0 with a background less than 0 30 amp 2 For kinetic analysis on graph paper plot the mean absorbance values for each X time points on the Y axis versus the time of each reaction minutes on the X axis Q Assay Characteristics g The CycLex Research Product CycLex Polo like kinase 2 Assay Inhibitor Screening Kit has
15. orking Solution y 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10 ash Buffer provided to 900 mL of ddH20 Mix well Store at 4 C for two weeks or 20 C for petem storage 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH20 to lyophilized Mix gently until dissolved The final concentration 1 25 mM Store the solution in small aliquots e g 100 uL at vial of 20X ATP provided e 20X ATP Solution should be 3 Prepare 20X DTT Solution by adding 0 5 mL of dd to the vial of 20X DTT provided lyophilized Mix gently until dissolved The final conc ion of the 20X DTT Solution should be 100 mM Store the solution in small aliquots e g 200 at 20 C 4 Prepare Kinase Reaction Buffer by mixing flloa reagents Ww 6 assays 10 assays 1 assay Kinase Buffer provided lt 9 0 mL 900 pL 90 uL 20X DTT Solution 72 0 5 mL 50 uL 5 uL 20X ATP Solution K 0 5 mL 50 uL 5 uL Total 10 mL 1000 uL 100 uL You will need 80 90 ne Kinase Reaction Buffer per assay well Mix well Discard any unused Kinase Regn Buffer after use Standard Assay 1 Remove the approp number of microtiter wells from the foil pouch and place them into the well holder keumey nused wells to the foil pouch refold seal with tape and store at 4 C 2 Prepare all Maples diluted with Kinase Buffer as needed All samples should be assayed in duplicate 0 O 3 Add R of individual purified PIk2
16. ow than 20 C oo 11 Add 100 uL of Stop Solution to each well in the sargprorder as the previously added Substrate Reagent O 450 540 nm Dual wavelengths of 450 550 o 595 nm can also be used Read the plate at 450 nm if only a single wavelength can be used ls must be read within 30 minutes of adding the Stop Solution O Note 1 Complete removal of liquid at caer is essential to good performance After the last wash remove any remaining Wash But r by aspirating or decanting Invert the plate and blot it against clean paper towels 74 Note 2 Reliable signals are obtained Khen either O D values do not exceed 0 3 units for the blank no enzyme control or 2 5 Gits for the 50 m units well of Plk2 positive control Cat 12 Measure absorbance in each well using a spectraphotometric plate reader at dual wavelengths of Ri CY E1183 Note 3 If the microplate reade ot capable of reading absorbance greater than the absorbance of the PIk2 positive cont rform a second reading at 405 nm A new O D values measured at 405 nm is used MK determine Plk2 activity of off scale samples The readings at 405 nm should not a on scale readings at 450 nm Kinetic Assays aS 1 Remove the aggropriate number of microtiter wells from the foil pouch and place them into the well holder Retgy ny unused wells to the foil pouch refold seal with tape and store at 4 C 2 Prepare samples diluted with Kinase Buffer as needed 3 A PuL
17. pose of tetra methylbenzidine crn Hain solutions in compliance with local regulations e Avoid contact with Substrate Solutigg hich contains hydrogen peroxide e Avoid contact with Stop Solutiongyfich contains Sulfuric Acid e In case of contact with the BPP Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention en necessary Biological samples gt contaminated with infectious agents Do not ingest expose to open wounds or breath osols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfyric Acid is a strong acid Wear disposable gloves and eye protection when h amp aling Stop Solution a Roa CY 1183 6 Version 150629 o AO Polo like kinase 2 Assay Inhibitor Screening Kit P 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Detailed Protocol 3 The CycLex Polo like kinase 2 Assay Inhibitor Screening Kit is provided with removablg Fips of A N wells so the assay can be carried out on separate occasions using only the number of strips ired for the particular determination Since conditions may vary running an aliquot of the ap iate Plk2 positive control Cat CY E1183 separately available from CycLex should be include yjn each assay Disposable pipette tips and reagent troughs should be used for all transfers to avoid Noran of reagents or samples Y Q Preparation of W
18. sample to the well of the assay plate on ice Duplicate wells contafng 50 m units 10 uL of Plk2 positive control Cat CY E1183 should be included in each a as a positive control for phosphorylation aK kein the kinase reaction by addition of 90 uL Kinase Reaction Buffer per well cover with plate amp sealer or lid and incubate at 30 C for 60 minutes A Roa CY 1183 T Version 150629 o AO A Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures S Polo like kinase 2 Assay Inhibitor Screening Kit lt 5 Wash wells five times with Wash Buffer making sure each well is filled completely Re e residual Wash Buffer by gentle tapping or aspiration 6 Pipette 100 uL of Anti Phospho Threonine Polyclonal Antibody into each well cov th plate sealer or lid and incubate at room temperature ca 25 C for 60 minutes Discar y unused antibody after use 7 Wash wells five times as same as in step 5 S Pipette 100 uL of HRP conjugated Anti rabbit IgG into each well covey plate sealer or lid and incubate at room temperature ca 25 C for 60 minutes Discard apay nused conjugate after use aS 9 Wash wells five times as same as in step 5 gt 10 Add 100 uL of Substrate Reagent to each well and incubate om temperature ca 25 C for 5 15 minutes Appropriate incubation time may vary and i ation time can be extended up to 20 minutes if the reaction temperature is bel
19. semi quantitative immunoassay for Plk2 activity Plates are pre coated with ubstrate corresponding to recombinant Plk2 substrate which contains threonine residues that ia ea phosphorylated by Plk2 The detector antibody specifically detects only the phospho threonine residue on Plk2 substrate The CycLex Polo like kinase 2 Assay Inhibi may be used to study the kinetics of a purified Plk2 as well as to screening PIk2 inhibi perform the test the sample is diluted in Kinase Buffer pipetted into the and allowed to phosphorylate the bound substrate following the addition of Mg and The amount of phosphorylated substrate is measured by binding it with a PPT 08 a anti phos Jho threonine polyclonal antibody followed by binding with horseradish peroxidase conjugated Qorabbit IgG which then catalyzes the conversion of the chromogenic substrate tetra methylbenaidin TMB from a colorless solution to a blue solution or yellow after the addition of stopping reag p The color is quantitated by spectrophotometry and reflects the relative amount of Plk2 activity inge sample For kinetic analysis the Plk2 containing sample is added to the wells in a similar fashion ghd at varying times the reaction is stopped by the addition of the chelator sodium ethylenediaminet Mrcetate EDTA and the amount of phosphorylated substrate determined as before AN lt Summary of Procedure 72 Q Add 100 uL of sample to the wet or activator To
20. t the ATP must be stored at 20 C Coate say plates should be stored in the original foil bag sealed by the zip lock and containing a desictant pack For rsa only not for use in diagnostic or therapeutic procedures Q S 72 S Polo like kinase 2 Assay Inhibitor Screening Kit Pe GA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Example of Test Results 3 Fig 1 Dose dependency of recombinant PIk2 enzyme reaction aS Pik2 dose dependency A450 0 25 50 75 100 Plk2 dose mUnit ST Fig 2 Dose dependency of ATP SS A Dose dependency of ATP 3 0 2 9 2 0 N als 1 0 0 5 Q oo Qe 0 20 40 60 80 100 ATP conc uM n uM c Roa CY 1183 13 Version 150629 S C RS 4 Polo like kinase 2 Assay Inhibitor Screening Kit ge cle User s Manual A gt 4 y For Research Use Only Not for use in diagnostic procedures Fig 3 Km for ATP recombinant PIk2 l Km for ATP N 1200 1000 S 800 V 600 y 10 467x 79 938 in 400 R 0 9854 Km for ATP 7 6 uM 200 0 O 10 20 30 40 50 60 70 80 90 100 ATP conc uM Fig 4 Effect of Polo like Kinase specific ae Polo like Kinase Inhibitor I Calbiochem on PIk2 activity wS S O Inhibition by Plk Inhibitor I 2 0 1 5 lt 10 0 5 l l SY o0 Q
21. ty the weakened as compared with Solvent control The high level of A450 is not ob control usually A450 lt 0 5 el of A450 is d in Inhibitor Solvent Inhibitor Assay reagents Test sample contd control Kinase Reaction buffer 80 uL sq 80 uL 10X Inhibitor or equivalent 10 pL oO Vehicle for Inhibitor amp uL 10X PIk Inhibitor I 250 uM amp 10 uL PIk2 positive control 5 m units uL or Purified enzyme sample Wy iy Ipe Opr Calbiochem Cat 528282 See Page 4 section Materials EA not Provided Plk2 positive control Cat CY E1183 See Page 5 section a Required but not Provided 1 Following the above table add the Reagents town well of the microplate Finally initiate reaction by adding 10 uL of PIk2 positive control units uL to each well and mixing thoroughly at room temperature Cover with plate sealer orgy and incubate at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 12 q6 e 7 8 Note Although we suggest to condifes experiments as outlined in the table above the optimal experimental conditions will v y depending on the parameters being investigated and must be determined by the individual Cr Especially appropriate amount of Plk2 positive control must be determined by titration of 1k2 positive control and setting the amount which shows OD value does not exceed platea e in dose response curve OPTIMAL E IMENTAL CONDITIONS WILL VAR
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