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CY-1176

1. Km of ATP s 5000 0 amp 4000 0 9 30000 y 34 098x 6 3458 R 0 9996 w 2000 0 Km of ATP 0 19uM 1000 0 0 0 0 20 40 60 80 100 ATP conc uM Rs Fig 6 Effect of NaCl on PIk3 activity lt xO Effect of NaCl 2 0 15 Q 10 s lt 0 5 0 0 P 0 0 0 1 0 2 0 3 04 05 O NaCl conc M amp a 2 Roa CY 1176 13 Version 140318 amp lt C A S Polo like kinase 3 Assay Inhibitor Screening Kit e cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 References X 1 Donohue P J Alberts G F Guo Y and Winkles J A Identification by targeted differe display of an immediate early gene encoding a putative serine threonine kinase J Biol Chw 270 10351 10357 1995 2 Simmons D L Neel B G Stevens R Evett G and Erikson R L Identification o early growth responsive gene encoding a novel putative protein kinase Mol Cell Biol 12614169 1992 3 Dai W Li Y Ouyang B Pan H Reissmann P Li J Wiest J Stambrook Gluckman J L Noffsinger A and Bejarano P PRK a cell cycle gene localized to 8p21 ig downregulated in head and neck cancer Genes Chromosomes Cancer 27 332 336 2000 4 Li B Ouyang B Pan H Reissmann P T Slamon D J Arceci R Qe and Dai W Prk a cytokine inducible human protein serine threon
2. s Manual 3 For Research Use Only Not for use in diagnostic procedures 4 Example of Test Results 3 Fig l Dose dependency of recombinant PIk3 enzyme reaction PIk3 dose dependency Si 2 00 1 50 e ATP amp ATP 2 1 00 lt 0 50 0 00 0 25 50 75 PIk3 dose mUnit Ley Fig 2 Time course of recombinant PIk3 enzyme Fiction O Timecourse 2 0 A450 Oo Q 0 20 40 60 80 100 120 140 Q o Roa CY 1176 11 Version 140318 o c Reactiontime min A Polo like kinase 3 Assay Inhibitor Screening Kit f ycLex User s Manual rey For Research Use Only Not for use in diagnostic procedures 4 Inhibition by Staurosporine N 120 0 74 Fig 3 Effect of broad spectrum kinase inhibitor staurosporine on activity of recombinant P1k3 77 S gt o oO N d Relative Intensity control e2 o d 0 10 20 30 40 Staurosporine conc uM Fig 4 Dose dependency of ATP SS Dose dependency of ATP 2 00 1 50 ip 1 00 lt 0 50 0 00 G 0 10 20 30 40 50 S ATP cone uM s oe Roa CY 1176 12 Version 140318 lt C Polo like kinase 3 Assay Inhibitor Screening Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Km for ATP S
3. ONLY AS A GUIDELINE THE Polo like kinase 3 Assay Inhibitor Screening Kit P 4 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Evaluation of Results 3 1 Average the absorbance values for the PIk3 sample duplicates positive control and all ex Rhental sample duplicate values when applicable When the PIk3 positive control 8 m un amp ssay is included as an internal control for the phosphorylation reaction the absorbance val hould be greater than 1 0 with a background less than 0 2 amp 2 For screening of purification chromatography fractions of recombinant PIk3 on paper plot the mean absorbance values for each of the samples on the Y axis versus the fr n number on the X axis to determine the location of the eluted purified P1k3 R 3 For kinetic analysis on graph paper plot the mean absorbance values f h of the time points on the Y axis versus the time of each reaction minutes on the X axis nmn Assay Characteristics gt The CycLex Research Product CycLex Polo like kinase 3 As nhibitor Screening Kit has been shown to detect the activity of PIk3 in column fractions of re gmbinant Plk3 The assay shows good linearity of sample response The assay may be used to follow due purification of recombinant PIk3 Troubleshootin yy 1 The Plk3 positive control should be run in dupe using the protocol described in the Detailed Protocol Incubation times or temperatu
4. 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 Plk3 substrate 1 as augers of PIk3 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 2 RY Kinase Buffer One bottle containing 20 mL of 1X buffer used for Kinase Re Beion Buffer and sample dilution S 20X ATP One vial of lyophilized ATP Naz salt RG HRP conjugated Detection Antibody One vial containing 12 HRP horseradish peroxidase conjugated anti phospho PIk3 substrate 1 monoclonal ae Ready to use Substrate Reagent One bottle containing 20 mL of the chrgnogenic substrate tetra methylbenzidine TMB Ready to use rA Stop Solution One bottle supplied ready to use containing20 mL of 1 N H2501 Materials Required but not Provi e PIK3 positive control Available from Cycl Rat CY E1176 One vial contains 1 6 units 200 uL 8 m units uL Plk3 enzyme Positive contgol should be added to the first well at ca 8 m units well Unused PIk3 enzyme should be stored ig iguots at below 70 C e 10X Staurosporine 100 uM Sauron is available from Sigma Cat S 4400 10 mM stock solution DMSO diluted 1 100 in Kifa e Buffer e Pipettors 2 20 uL 20 200 uL and P 1000 uL precision pipettors with disposable tips e Precision repeating pipettor e Wash bottle or m
5. in th test chemicals correctly it is necessary to conduct the control experiment of Solvent control at leastg once for every experiment and Inhibitor control at least once for the first experiment in addition to Gest sample as indicated in the following table When test chemicals cause an inhibitory effect on activity the level of A450 is weakened as compared with Solvent control Recommendations Assay reagents Texeefiple Solvent control ee Kinase Reaction Buffer o uL 80 uL 80 uL 10X Inhibitor or equivalent 10 pL Solvent for Inhibitor l 10 uL 10X Staurosporine 100 uM 74 10 uL PIk3 Positive Control 0 8 m mig 10 uL 10 uL 10 uL or your enzyme fraction u u 10X Staurosporine 100 uM See paste section Materials Required but not Provided Cat CY E1176 See page 4 eG aterials Required but not Provided 1 Following the above table d the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of Diftted Plk3 positive control to each well and mixing thoroughly at room temperature Cover w 2 Follow the TA Assay steps te sealer Incubate at 30 C for 30 minutes 5 10 page 6 7 Note Although ve suggest to conduct experiments as outlined in the table above the optimal experim deter al conditions will vary depending on the parameters being investigated and must be d by the individual user Especially
6. is efficiently phosphorylated by PIk3 in vi Applications of this kit wai 1 Screening inhibito ctivators of Plk3 2 Detecting the e of pharmacological agents on PIk3 activity This assay kit is f research use only and not for use in diagnostic or therapeutic procedures ww Q O Q Roa CY 1176 1 Version 140318 o AO Polo like kinase 3 Assay Inhibitor Screening Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 The polo like kinases Plks are a family of conserved serine threonine kinases that play a oR role in the normal progression of cells through mitosis The Plk3 serine threonine kinase is a malian member of this family In contract to Plkl overexpression of PIk3 in mammalian ce uppresses proliferation and inhibits colony formation Subsequent analysis demonstrated that Ov xpression of PIk3 induces chromatin condensation and apoptosis One difference between Plk1 and ther two Plks is that both P k2 and PIk3 were originally identified as immediate early genes 1 2 ereas Plk1 does not share this characteristic The Plks have been implicated in the genesis or progr n of tumors PIk3 has been suggested as a candidate tumor suppressor 3 and its expression is dawn regulated or absent in lung carcinomas 4 and squamous cell carcinomas of the head and neck 3 PIk3 contributes to regulation of M phase of the cell cycle 5 Plk3 phyA int
7. Polo like kinase 3 Assay Inhibitor Screening Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 gt Non Radioisotopic Kit for Measuring PIk3 activity l CycLex Polo like kinase 3 Assays Screening Kit Cat CY 1176 2 gt Intended USse cceeeeecccccececccsseeeseeseseesecees 1 ne Be sat nsatsinanigayonmnysaneiocsiciassadsaepiaaeeeisinen 1 a Introduction seeseeeeeeeeeseeeeeresrerseseresrreresrreees 2 Yon Principle Of the Assay 3 gt Materials Provided ceccceeeseeeeeeeeeeees 4 o Materials Required but not Provided 4 Precautions and Recommendations 5 4 Detailed Protocol cccccccccceceecessseeeseeeeees 6 9 amp Evaluation of Results 10 4 Assay Characteristics reee 10 gt Troubleshooting s sscaenesssscsectsnndoonsnuceabnonnnsaicn 10 Reagent Stability we Example of Test Results cee eeeeeeeeees or References cccccssssessecsesecssesecsessessesecseeseeseg 4 Related Products cccccceeeeeeeeee ees Q 14 Intended Use amp The CycLex Research Product Cy ex Polo like kinase 3 Assay Inhibitor Screening Kit designed to measure the activities of purified ae for the rapid and sensitive evaluation of inhibitors or activators The sequence specific phosphoserigie monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphosesy residue in recombinant Plk3 substrate 1 which
8. appropriate amount of Plk3 positive control must be detewMined by titration of the Plk3 positive control and setting the amount which shows OD value g ot exceed plateau range in dose response curve NO THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE 2 PARAMETERS BEING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDUAL USER NO WARRANTY OR GUARANTEE OF PERFORMANCE Version 140318 A gt g Polo like kinase 3 Assay Inhibitor Screening Kit f cle User s Manual A v y x For Research Use Only Not for use in diagnostic procedures amp gt USING THESE PROCEDURES IS MADE OR IMPLIED O Special considerations when measuring precise PIk3 activity In order to measure the activity of PIk3 correctly it is necessary to conduct the control exp nt of Inhibitor control at least once for every experiment and ATP minus control at least once e first experiment in addition to No enzyme control as indicated in the following table Althou e level of A450 increases in Test sample when Plk3 enzyme activity is in the sample the high l yel of A450 is not observed in Inhibitor control ATP minus control and No enzyme control N Assay reacents Test Inhibitor ATP minus Pee No enzyme y reag Sample control control orrtrol control Kinase Reaction Buffer 90 uL 80 uL 700 pL 90 uL Kinase Buffer pr
9. ate on ice D ate wells containing 8 m units 10 uL PIk3 positive control Cat CY E1176 3 To assay partiall Gaited recombinant Plk3 add 10 uL of each fraction to the wells of the assay should be ng in each assay as a positive control for phosphorylation 4 Begin the se reaction by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer ncubate at 30 C for 60 minutes 5 W ells five times with Wash Buffer making sure each well is filled completely Remove r s ual Wash Buffer by gentle tapping or aspiration Ken 100 uL of HRP conjugated Detection Antibody into each well cover with a plate sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate Polo like kinase 3 Assay Inhibitor Screening Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 7 Wash wells five times with Wash Buffer making sure each well is filled completely Re e residual Wash Buffer by gentle tapping or aspiration 8 Add 100 uL of Substrate Reagent to each well and incubate at room temperature 5 15 minutes 9 Add 100 uL of Stop Solution to each well in the same order as the previous tea Substrate Reagent gt 10 Measure absorbance in each well using a spectrophotometric plate reade aaa wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be u ead the plate at 450 nm if only a single wavelength can be used Wells must be read w
10. bitor Screening Kit uses a peroxidase coupled anti phospho Plk3 substrate 1 monoclonal antibody as a reporter molecule in a Jo wee AISA format This assay provides a non isotopic sensitive and specific method to detect Plk3 acti Ss o oe Roa CY 1176 2 Version 140318 o AO Polo like kinase 3 Assay Inhibitor Screening Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Principle of the Assay 3 The CycLex Research Product CycLex Polo like kinase 3 Assay Inhibitor Screening it is a single site semi quantitative immunoassay for Plk3 activity Plates are pre coated with ubstrate corresponding to recombinant Plk3 substrate 1 which contains a serine residue that is ea aaa by Plk3 Polo like kinase 3 amp The detector antibody specifically detects only the phosphorylated form of Plk3 strate 1 The CycLex Research Product CycLex Polo like kinase 3 Assay Inhibitor Screeninggkit can be used to study the kinetics of a purified or partially purified Plk3 as well as to screening t kinases inhibitor To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substrate in the presence of Mg and ATP The afgpunt of phosphorylated substrate is measured by binding it with a horseradish peroxidase jugate of KS 3H4 an anti phospho PIk3 substrate 1 specific antibody which then catakyz 8 the conv
11. cular determination Since experimental conditi N nay vary an aliquot of the Plk3 Cat CY E1176 available separately from CycLex should be ig jJuded in each assay aS a positive control Disposable pipette tips and reagent troughs should be ete all liquid transfers to avoid cross contamination of reagents or samples Y Preparation of Working 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 Kinase Reaction Buffer by mixing following reagis 96 SA 10 assays 1 assay Kinase Buffer provided 9 5 ny 950 uL 95 uL 20X ATP Solution AL 50 uL 5 uL Total x2 mL 1000 uL 100 uL You will need 80 90 uL of Kinase ion Buffer per assay well Mix well Discard any unused Kinase Reaction Buffer after use Standard Assay amp 1 Remove the appropriate numbe Pf microtiter wells from the foil pouch and place them into the well holder Return any unused to the foil pouch refold seal with tape and store at 4 C 2 Prepare all samples ated with Kinase Buffer as needed All samples should be assayed in duplicate pl
12. en approval from CycLex Co Ltd To inquire about licensing for such gommercial use please contact us via email PRODUCED BY rA Roa CY 1176 14 Version 140318 o AO
13. eracts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 19 1 amp 6 suggesting that the role of PIk3 in mitosis is mediated at least in part through direct regulation AESC In response to DNA damage the kinase activity of PIk3 was rapidly increased in an ATM ndent manner whereas that of Plk1 was markedly inhibited Co immunoprecipitation and pull doQr assays demonstrated that PIk3 physically interacted with p53 and that this interaction was enhhanc apon DNA damage Taken together these studies indicate that Plk3 functionally links DNA damage ll cycle arrest and apoptosis via the p53 pathway 7 lt amp The protocol generally regarded as most sensitive Gy the quantitative measurement of PIk3 activity involves incubation of the Plk3 sample with subsp vier a natural or synthetic polypeptide such as Cdc25C S191 peptide RRRDQAEEISDELMEF if the presence of Mg and P labeled ATP The Measurement of PIk3 activity reaction is terminated by spotting a sample phosphocellulose P81 filter paper disc followed by washing extensively to remove unincorporated radiolabel and the incorporated radioactivity on P81 filter is counted While sensitive this method 1 her intensive generates hazardous radioactive waste and depends on a radioisotope of short half It is particularly unsuitable when kinase assays are only performed on an infrequent basis Th cLex Research Product CycLex Polo like kinase 3 Assay Inhi
14. ersion of the chromogenic substrate tetra methylbenzidine TMB from a colorless tion to a blue solution or yellow after the addition of stopping reagent The color is quantified kspectrophotometry and reflects the relative amount of Plk3 activity in the sample For kinetic analis the sample containing PIk3 is added to the wells in a similar fashion and at varying times the r ion is stopped by the addition of a chelator sodium ethylenediaminetetraacetate EDTA and hount of phosphorylated substrate determined as before The CycLex Research Product CycLex Polo like kinaga 3 Assay Inhibitor Screening Kit is designed to accurately determine the presence and relative gmount of Plk3 kinase activity to determine non isotopic kinetic analysis of PIk3 activity amp Summary of Procedure aS Add 100 uL of reactii to the wells 4 ea ubate for 60 min at 30 C Wash t ells Add 100 a HRP conjugated anti phosphorylated form specific antibody 4 Incubate for 60 min at room temp Q Wash the wells a Qia 100 uL of Substrate Reagent t Pg Add 100 uL of Stop Solution O t Q Measure absorbance at 450 nm Qe gL amp Roa CY 1176 3 Version 140318 o AO A S Polo like kinase 3 Assay Inhibitor Screening Kit Pe r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Materials Provided 3 All samples and standards should be assayed in duplicate The following components are ua
15. ine kinase whose expressio appears to be down regulated in lung carcinomas J Biol Chem 271 19402 19408 6 5 Bin Ouyang Huigi Pan Luo Lu Jian Li Peter Stambrook Bo Li a ei Dai Human Prk Is a Conserved Protein Serine Threonine Kinase Involved in Regulating Phase Functions J Biol Chem 272 28646 28651 1997 6 Bahassi el M Hennigan RF Myer DL Stambrook PJ Cd phosphorylation on serine 191 by PIk3 promotes its nuclear translocation Oncogene 23 15 2 58 63 2004 7 Plk3 Functionally Links DNA Damage to Cell Cycle Arreg gnd Apoptosis at Least in Part via the p53 Pathway Suqing Xie Huiyun Wu Qi Wang John Co ell Intisar Husain Chris Conn Peter Stambrook Meena Jhanwar Uniyal and Wei Dai J a 276 43305 43312 2001 Related Products AY Plk1 Positive control Cat CY E1163 xO Plk2 Positive control Cat CY E1183 PIk3 Positive control Cat pre a CycLex Polo like kinase 1 Assay Tnhiby r Screening Kit Cat CY 1163 CycLex Polo like kinase 2 Assay Inliibitor Screening Kit Cat CY 1183 CycLex Polo like kinase 3 i E Screening Kit Cat CY 1176 Y amp CycLex Co Ltd WwW 1063 103 Terasawaok Ina Nagano 396 0 Japan Fax 81 265 76 9018 e mail info X CO j URL btn gs cyclex co jp CycLe DruLex products are supplied for research use only CycLex CircuLex products and compon nts thereof may not be resold modified for resale or used to manufacture commercial prodgets without prior writt
16. is minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good ormance After the last wash remove any remaining Wash Buffer by aspirating or de ng Invert the plate and blot it against clean paper towels Y Note 2 Reliable signals are obtained when either O D valu a exceed 0 25 units for the blank no enzyme control or 2 5 units for the PIk3 posigfeeonto Note 3 If the microplate reader is not capable of readi sorbance greater than the absorbance of the Weel positive control perform a second reading at 405 nm A new O D values measured at 405 nm is used to determine Plk3 actiyg f off scale samples The readings at 405 nm should not replace the on scale readings W450 nm at 1 Remove the appropriate number of m iter wells from the foil pouch and place them into the well holder Return any unused wells to Soil pouch refold seal with tape and store at 4 C Kinetic Assay N Prepare all samples diluted pW Kinase Buffer as needed All samples should be assayed in duplicate Y w To assay partially prion Plk3 add 10 uL of each fraction to the wells of the assay plate on ice Duplicate Wells containing 8 m units 10 uL Plk3 positive control Cat CY E1176 should be included S assay as a positive control for phosphorylation N 4 Begin kinase re by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervals sugg interval is 5 minutes but should be i
17. ndividually determined for each system After the finaggddition incubate at 30 C for 30 minutes 5 Stop the r ption by flicking out the contents Alternatively the reaction may be terminated by the addition 150 uL 0 1 M Na EDTA pH 8 0 to each well 6 Wome five times with Wash Buffer making sure each well is filled completely Remove ual Wash Buffer by gentle tapping or aspiration Kre 100 uL of HRP conjugated Detection Antibody KS 3H4 into each well cover with a plate eA sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate after use Rea CY 1176 7 Version 140318 o AO yclex Polo like kinase 3 Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnostic procedures 8 Wash wells five times as same as in step 6 9 Add 100 uL of Substrate Reagent to each well and incubate at room temperature 5 15 minutes 10 add 100 uL of Stop Solution to each well in the same order as the previously tea Substrate Reagent 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be u ead the plate at 450 nm if only a single wavelength can be used Wells must be read wit minutes of adding the 11 Measure absorbance in each well using a spectrophotometric plate aaa wavelengths of Stop Solution ko Q Special considerations when screening activators or inhibitors Y In order to estimate the inhibitory effect on Plk3 activity
18. ovided 90 uL mY 10X Staurosporine 100 uM 10 uL gt Your enzyme fraction 10 uL 10 uL my Pik3 Positive Control 0 8 m unit uL j amp i 10 pL Buffer 10 uL 10X Staurosporine 100 uM See page 4 section Materials Requiregp not Provided Cat CY E1176 See page 4 section Materials Required but not A ded 1 Following the above table add the Reagents to ead well of the microplate Finally initiate the reaction by adding 10 uL of Your enzyme fractio Sr Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer Inca at 30 C for 30 minutes 2 Follow the Standard Assay steps 5 10 pagal experimental conditions will vary nding on the parameters being investigated and must be determined by the individual user E8pecially appropriate amount of PIk3 positive control must be determined by titration of the Pes positive control and setting the amount which shows OD value does not exceed plateau ranga dose response curve Note Although we suggest to ing GSeriments as outlined in the table above the optimal OPTIMAL EXP NTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDUAL R NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESRY OCEDURES IS MADE OR IMPLIED O nw amp a Roa CY 1176 9 Version 140318 o c NOTE THE ABOVE NENTA ARE INTENDED
19. r chemicals Care should be taken to avoid direct contact with these reagents KS Do not mouth pipet or ingest any of the reagents aS e Do not smoke eat or drink when performi e assay or in areas where samples or reagents are handled lt e Dispose of tetra methylbenzidine cron 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 uling Stop Solution a Qe amp eo Roa CY 1176 5 Version 140318 o AO A Roa CY 1176 6 Version 140318 ge A S Polo like kinase 3 Assay Inhibitor Screening Kit Pe r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Detailed Protocol 3 The CycLex Research Product CycLex Polo like kinase 3 Assay Inhibitor Scree ni it is provided with removable strips of wells so the assay can be carried out on separate occasiongsing only the number of strips required for the parti
20. res sigM ficantly different from those specified may give erroneous results w 2 The reaction curve is nearly a straight li negithe kinetics of the assay is of the first order Variations in the protocol can lead to non linearity o curve as can assay kinetics that are other than first order For a non linear curve point to point eGtadratic curve fit methods should be used 3 Poor duplicates accompanied by ated values for wells containing no sample indicate insufficient washing If all instructions in t etailed Protocol were followed accurately such results indicate a need for washer maintenance Qy 4 Overall low signal may Rate 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 N Reagent Stability Se All of the r nts included in the CycLex Research Product CycLex Polo like kinase 3 Assay Inhibitor S ing Kit have been tested for stability Reagents should not be used beyond the stated expiration dat 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 desi t pack Fogfeearch use only not for use in diagnostic or therapeutic procedures e Rom CY 1176 10 Version 140318 o AO S Polo like kinase 3 Assay Inhibitor Screening Kit 9 A ycLex User
21. ultichannel digpenser for plate washing e Microcentrifuge and tubes mple preparation e Vortex mixer e Plate reader capable of yeasuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengt 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength o nm which will give a somewhat higher reading e 500 or 1000 mL g ated cylinder e Reagent reservoir Deionized wat the highest quality Disposable p towels a Qe amp eo Roa CY 1176 4 Version 140318 ge ww S Polo like kinase 3 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 ATP at 20 C in aliquots Store all other components at 4 C Do not expose nfernts 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 othe

CY-1176

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