CY-1356 Protein Phosphatase Cdi1/KAP Fluorometric Assay Kit
Contents
1. Protein Tyrosine Phosphatase PTPRD 2nd Catalytic Domain Cat CY E1307 Protein Tyrosine Phosphatase PTPRE 1st Catalytic Domain Cat CY E1308 Protein Tyrosine Phosphatase PTPRF 1st Catalytic Domain Cat CY E1310 Protein Tyrosine Phosphatase PTPRK 1st Catalytic Domain Cat CY E1316 Protein Tyrosine Phosphatase PTPRQ Cat CY E1323 Protein Tyrosine Phosphatase PTP4A2 Cat CY E1341 Recombinant Cdc25A Catalytic domain Cat CY E1352 Recombinant Cdc25B Catalytic domain Cat CY E1353 Recombinant Cdc25C Catalytic domain Cat CY E1354 Recombinant Cdil KAP Cat CY E1356 Protein Phosphatase PPS Cat CY E1359 Protein Tyrosine Phosphatase PTPN3 PTPH1 Cat CY E1360 Protein Tyrosine Phosphatase PTPN6 SHP 1 Cat CY E1363 Protein Tyrosine Phosphatase PTPN7 HePTP Cat CY E1364 Cat CY 1356 11 Version 120420 pry Protein Phosphatase Cdil KAP Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Protein Tyrosine Phosphatase PTPN8 PTPN22 Cat CY E1365 Protein Tyrosine Phosphatase PTPN9 MEG2 Cat CY E1366 Protein Tyrosine Phosphatase PTPN11 SHP 2 Cat CY E1367 Protein Tyrosine Phosphatase PTPN12 PTP PEST Cat CY E1368 Protein Tyrosine Phosphatase PTPN13 FAP 1 Cat CY E1369 Protein Tyrosine Phosphatase PTPN14 PEZ Cat CY E1370 Protein Tyrosine Phosphatase PTPN21 PTPD1 Cat CY E1372 Protein Phosphatase DUSP1 MKP 1 Cat CY E2. and emission at 510 540 nm v 4 Measure and calculate the rate of reaction while the reaction velocity remains constant Caution and Significance e All samples and Recombinant Cdil KAP should be assayed in duplicate e Use of a microtiter plate shaker is recommended for complete mixing e If the test compounds or samples themselves emit fluorescence at excitation wavelength 482 502 nm and fluorescence wavelength 510 540 nm the test aSsay cannot be evaluated correctly Cat CY 1356 6 Version 120420 pry Protein Phosphatase Cdil KAP Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Analysis of Inhibitor Effect 1 Intensity 1 Run reactions with test compounds and Vehicle as described in the Detailed Protocol 2 Subtract fluorescence intensity of No Enzyme Control from all experimental samples L st Samples and Vehicle Control 3 Calculate the Intensity Fluorescence Intensity of Test Sample Intensity X 100 Fluorescence Intensity of Vehicle Control Note This Intensity is a rough value of enzyme activity or inhibition For greater accuracy plot a standard curve of Cdil KAP for each new set of reactions and estimate the Activity see below Fig 1 Cdil KAP Inhibition Curve by Na VO Na3VO4 Sodium Orthovanadate s 4 0 0 0001 0 001 0 01 0 1 1 10 100 1000 10000 Na3V
3. 1356 3 Version 120420 Protein Phosphatase Cdil KAP Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures 7 YCLEX NOTE THE FOLLOWING PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDUAL USER Detailed Protocol Preparation of Reagents Thaw the reagents at room temperature except Recombinant Cdil KAP and keep all reagents on ice until use Use them only after they are completely thawed and mixed 1 Prepare 10X Cdil Inhibitor by adding 5 uL of the 100X Cdil Inhibitor provided to 45 uL of distilled deionized water Mix well Discard any unused 10X Cdil Inhibitor after use 2 Prepare Assay Mixture by adding 5 uL of the 10X Assay Buffer provided and 5 uL of the 10X Fluoro Phospho Substrate provided to 30 uL of distilled deionized water per one assay Mix well Assay Mixture Assay reagents l assay 8 assays 16 assays 32 assays 48 assays Distilled water 30 uL 240 uL 480 uL 960 nL 1 440 uL 10X Assay Buffer 5 uL 40 uL 80 uL 160 uL 240 uL 10X OMFP 5 nL 40 uL 80 uL 160 uL 240 uL Total volume of Assay Mixture 40 uL 320 uL 640 uL 1 240 uL 1 920 uL Cat CY 1356 4 Version 120420 Protein Phosphatase Cdil KAP Fluorometric Assay Kit 4 gt ycLex User s Manual For Research Use Only Not for use
4. 1373 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 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 1356 12 Version 120420
5. O4 conc uM Cat CY 1356 T Version 120420 pry Protein Phosphatase Cdil KAP Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Analysis of Enzyme Activity Cdil KAP Standard Curve and Activity Dilute the 10X Assay buffer 1 10 with distilled water to make 1X Assay Buffer Make serial dilutions of Recombinant Cdil KAP with 1X Assay Buffer ex 100 50 25 12 5 6 25 3 13 and 0 3 Run reactions with Vehicle and serial dilutions of Recombinant Cdil KAP as described in the Detailed Protocol 4 Plot standard curve data dose dependent curve data as fluorescence intensity at 510 540 nm versus dose of Cdil KAP ng assay 5 Obtain a line fit to the data using appropriate calculations 6 Use the slope and Y intercept to calculate the amount of Cdil KAP activity for the experimental data P Fig 2 Dose Dependency of Recombinant Cdil KAP 3 000 000 2 500 000 2 000 000 1 500 000 a t19 Ced 19 N 19 w ww C e pd o 1 000 000 500 000 0 1 dilution ratio Cat CY 1356 8 Version 120420 pry Protein Phosphatase Cdil KAP Fluorometric Assay Kit i ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Analysis of Kinetics Time Course Curve 1 Run reactions as described in the Detailed Protocol 2 Subtract fluorescence intensity at the 0 ti
6. P Human 0 4 pg L 550 uL x4 70 C 100X Cdil Inhibitor 100 uM Na VO in H2O 100 nlx Below 20 C Stop Solution 300 uL xt Below 20 C Instruction Manual 1 Room temp Materials Required but not Provided e Microtiter plate suitable for use with a fluorometric plate reader Fluorometric plate reader or microtiter plate fluorometer Use a fluorescence microplate reader equipped with appropriate filters OMFP has excitation emission maxima of approximately 485 525 nm We have found that standard filters for blue fluorescent dyes e g excitation 485 12 5 nm emission 525 20 nm can be used to detect OMFP Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips e Multi channel pipette e Microtiter plate shaker e Distilled water DW or equivalent high quality water e Microcentrifuge and tubes for samplepreparation e Reagent reservoirs Ice bucket to keep reagents cold until use Precautions and Recommendations e Upon receipt store the kit at 70 G Do not expose reagents to excessive light e Do not use kit components beyond the indicated kit expiration date e Rinse all detergentyresidue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits Do notmouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled Cat CY
7. ed water to each well and mixing thoroughly at room temperature 3 Incubate for 15 min or desired length of time at room temperature 4 Add 25 uL of G Stop Solution to each well of the microtiter plate and mix thoroughly 5 Measure fluorescence intensity using a microtiter plate fluorometer with excitation at 482 502 nm and emission at 510 540 nm 6 The efficacy of the Test compound is the difference in fluorescence intensity between Vehicle control and Test sample Note lf necessary it is possible to store the microtiter plate after adding Stop Solution for a few hours at 4 C The microtiter plate must be sealed to prevent evaporation and kept from excessive light Cat CY 1356 5 Version 120420 P Protein Phosphatase Cdil KAP Fluorometric Assay Kit ey cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Alternate procedure v 1 Following Table 1 above first add Assay mixture to microtiter plate wells Second add Test Compound or Vehicle of Test Compounds or 10X Cdil KAP Inhibitor to each well of the microtiter plate and mix well v 2 Initiate reactions by adding 5 uL of Recombinant Cdil KAP or distilled water to each well and mixing thoroughly at room temperature 3 Read fluorescence intensity for 20 to 30 minutes at 1 to 2 minute intervals using aaicrotiter plate fluorometer with excitation at 482 502 nm
8. ey Protein Phosphatase Cdil KAP Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fluorometric Assay Kit for Measuring Cdil KAP Phosphatase Activity CycLex Protein Phosphatase Cdil KAP Fluorometric Assay Kit 100 Assays Cat CY 1356 Intended US c scscsassessuscscensseecsennseeesnasoecns 1 PMOL ALS suet cucu E EEE T 1 Introduction isch ncaabaesdocsstapcaasistacaadibacess 2 Principle of the Assay ccceseeeeeees 2 Materials Provided 4 vissscsvisssaveassnccxeousseisess 3 Materials Required but not Provided 3 Precautions and Recommendation 3 4 Detailed Protocol ccccccccceesssseeeeesees 4 6 Evaluation of Results cccccceceerees 7 9 Troubleshooting socsscustsosdsoniscaniinsernesences 10 Reagent Stability syssessiieeccameinenssannawanes 10 RSTEEHOES c ssccssensteciesinssecsansdenvasenstecssanad 11 Related Products scvsmsssehinsssseatavzaadimmesouses 1 L2 Intended Use The CycLex Research product Protein Phosphatase Cdil KAP Fluorometric Assay Kit is a fluorometric and non radioactive assay designed to measure the activity of Cdil KAP protein phosphatase This 96 well assay is usefubfor screening inhibitors and modulators of Cdil KAP activity in HTS The kit includes all necessary components including recombinant human Cdil KAP full length for use in preinvestigational drug discovery assays This assay kit is for research use only and no
9. gulating a variety of signal transduction pathways that have a bearing on cancer 4 6 It was reported thatthe Cdil KAP gene is overexpressed in human breast and prostate cancer using differential screening and that breast and prostate malignancies are associated with high levels of KAP expression 7 Principle of the Assay The Protein Phosphatase Cdil KAP Fluorometric Assay Kit is based on an exclusive fluorescence substrate OMFP 3 0 methylfluorescein phosphate This homogenous assay kit is sensitive and convenient This method of measurement should raise the efficiency of inhibitor screening and biochemical analysis of this enzyme Summary of Procedure Mix 40 uL of Assay mixture and 5 uL of test compound in the wells Add 5 uL of Recombinant Cdil KAP v Incubate for 15 min at room temp Add 25k of Stop Solution v Measure fluorescence at 510 540 nm emission 482 502 nm excitation Cat CY 1356 2 Version 120420 wy Protein Phosphatase Cdil KAP Fluorometric Assay Kit y gt X 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 one hundred assays Components of Kit Components Quantity Storage 10X Assay Buffer 600 pLx1 Below 20 C 10X 3 o methy fluorescein phosphate OMFP 550 pL x1 Below 20 C Recombinant Cdil KA
10. in diagnostic procedures Assay Procedure In order to estimate the inhibitory effect on Cdil KAP activity by the test compounds correctly t is necessary to conduct the control experiment of Vehicle control at least once for every experiment and Inhibitor control at least once for the first experiment in addition to Test sample as indicatedun the Table 1 below When test chemicals cause an inhibitory effect on Cdil KAP activity the level of increase of fluorescence intensity is weakened as compared with Vehicle control Themincrease in fluorescence intensity is not observed in Inhibitor control 1 Following Table 1 below first add Assay mixture to microtiter plate wells Second add Test Compound or Vehicle of Test Compounds or 10X Cdil Inhibitor toyeach well of the microtiter plate and mix well Table 1 Reaction mixture Assay reacenis Test Vehicle 4 Inhibitor No Enzyme y reag Sample Control Control Control Assay Mixture 40 uL 40 pL 40 uL 40 uL Test Compound 5 pL gt Vehicle of Test Compounds Spb 5 uL 10X Cdil Inhibitor 5 uL Recombinant Cdil KAP 0 1ug uL SuL SuL SuL Distilled water 5 uL Total Volume of the Reaction mixturey 50 uL 50 uL 50 uL 50 uL 10X Cdil Inhibitor 10 uM Na3 3VO y SeePage 4 section Preparation of Reagents 2 Initiate reactions by adding 5 pL of Recombinant Cdil KAP or distill
11. me from all reaction time points 3 Plot fluorescence intensity at 510 540 nm versus reaction time 4 Determine the reaction time range in which the increase in fluorescence intensity at 510 540 nm is linear 5 Calculate activity Fluorescence Intensity of Test Sample Activity reaction velocity Reaction time min Note Usually the linear range is from 0 to 30 min This value i variable depending on reaction conditions and storage handling of the Recombinant Cdil KAP Decreasing the amount of Recombinant Cdil KAP in the assay may help to lengthen the time range Fig 3 Time Course Curve of Recombinant Cdil KAP 2 500 000 2 000 000 1 500 000 1 000 000 1O o 1O x sd eo ww C c S o o 500 000 30 0 Time min Cat CY 1356 9 Version 120420 pry Protein Phosphatase Cdil KAP Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The Recombinant Cdil KAP should be run in 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 kinetics of the assay is of the first ordersVariations 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
12. n Science 270 90 3 1995 4 Dem J M M A Stuckey M Saper and J E Dixon Form andfunction in protein dephosphorylation Cell 87 361 364 1996 5 Kinzler K W and B Vogelstein Landscaping the cancer terrain Science 280 1036 1037 1998 6 Parsons R Phosphatases and tumorigenesis Curr Opin Oncol 10 88 91 1998 7 Lee SW Reimer CL Fang L Iruela Arispe ML Aaronson SA Overexpression of kinase associated phosphatase KAP in breast and prostate cancer and inhibition of the transformed phenotype by antisense KAP expression Mol Cell Biol 20 1723 32 2000 Related Products CycLex Protein Tyrosine Phosphatase 1B PTP1B Fluorometric Assay Kit Cat CY 1350 CycLex T Cell Protein Tyrosine Phosphatase TC PTP Fluorometric Assay Kit Cat CY 1351 CycLex Protein Phosphatase Cdc25A Fluorometric Assay Kit Cat CY 1352 CycLex Protein Phosphatase Cdc25B Fluorometric Assay Kit Cat CY 1353 CycLex Protein Phosphatase Cdc25C Fluorometric Assay Kit Cat CY 1354 CycLex Protein Phosphatase Cdc25 Combo Fluorometric Assay Kit Cat CY 1355 CycLex Protein Phosphatase Cdil KAP Fluorometric Assay Kit Cat CY 1356 CycLex Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit Cat CY 1358 CycLex Protein Phosphatase DUSP1 MKP 1 Fluorometric Assay Kit Cat CY 1373 Protein Tyrosine Phosphatase PTPRA 1st Catalytic Domain Cat CY E1301 Protein Tyrosine Phosphatase PTPRA 2nd Catalytic Domain Cat CY E1302
13. or quadratic curve fit methods should be used 3 Poor duplicates accompanied by elevated values for wells containing no sample indicate inaccurate dispensing of assay reagents If all instructions in the Detailed Protocol wetewfollowed accurately such results indicate a need for multi channel pipette maintenance Reagent Stability All of the reagents included in the Protein Phosphatase Cdil KAP Fluorometric Assay Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt all kit reagents except Recombinant Cdil KAP should be stored at 20 C Recombinant Cdil KAP should be stored at 70 C After use return kit reagents to 20 C asjsoon as possible For research use only not for use in human diagnosticor therapeutic procedures Cat CY 1356 10 Version 120420 pry Protein Phosphatase Cdil KAP Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References Gyuris J Golemis E Chertkov H Brent R Cdil a human G1 and S phase protein phosphatase that associates with Cdk2 Cell 75 791 803 1993 2 Hannon G J Casso D Beach D KAP a dual specificity phosphatase that interacts with cyclin dependent kinases Proc Nat Acad Sci 91 1731 1735 1994 3 Poon RY and Hunter T Dephosphorylation of Cdk2 Thrl60 by the cyclin dependent kinase interacting phosphatase KAP in the absence of cycli
14. t for use in human diagnostic or therapeutic procedures Storage e Upon receipt storethe kit at 70 C e Don t expose reagents to excessive light AVOID REPEATED FREEZE THAW CYCLES OF Recombinant Cdil KAP Cat CY 1356 1 Version 120420 Protein Phosphatase Cdil KAP Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction A Cdk interacting protein called Cdil cyclin dependent kinase interactor 1 KAP kinase associated phosphatase was first identified as a novel G1 and S phase dual specificity phosphatase that associates with Cdk2 and or Cdc2 by the interaction trap a yeast genetic selection for interacting proteins 1 2 Using yeast two hybrid system Cdil KAP interacts with cyclin dependent kinases including human Cde2 Cdk2 and Cdk3 but not with Cdk4 In HeLa cells Cdil KAP is expressed atetheyG1 to S transition and the protein forms stable complexes with Cdk2 Further studies demonstrated that Cdil KAP binds to Cdk2 and dephosphorylates Thr160 when the associated cyclin subunit isydegraded or dissociated 3 It means that Cdil KAP may inactivate a Cdk2 or Cdc2 by remoying phosphates from the cyclin complexes and this may contribute to cell cycle control 2 However the physiological substrate s for tyrosine dephosphorylation of Cdil KAP has not yet been identified Phosphatases have also been shown to play an important role in re
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