CY-1358 Protein Phosphatase LMW-PTP/ACP1 Fluorometric Assay Kit
Contents
1. 1 500 000 1 000 000 Counts 485nm 535nm 500 000 0 4 0 6 Dilution Ratio t CY 1358 8 Version 120420 A 4 Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit Yelex 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 time 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 linear 5 Calculate activity Fluorescence Intensity of Test S Activity reaction velocity Reaction time mi Note Usually the linear range is from 0 to 30 min This value i conditions and storage handling of the Recombinant LMW P depending on reaction ecreasing the amount of e time range 3 000 000 2 500 000 2 000 000 1 500 000 Counts 485nm 535nm 1 000 000 500 000 10 15 Time Min tH CY 1358 Version 120420 P Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit 4 c ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The Recombinant LMW PTP ACP1 should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from2. 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 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 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 LMW PTP ACP Fluorometric Assay Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt should be stored at 70 C Avoid repeated freeze thaw cycles of Recombinant LMW PTP ACP1 After use return kit reagents to 70 assoomias possible For research use only not for use in human diagnostic or therapeutic procedures References 1 Dissing J Johnsen AH Sensabaugh GF Human red cell acid phosphatase ACP1 The amino acid sequence of the two isozymes Bf and Bsgencoded by the ACP1 B allele J Biol Chem 1991 Nov 5 266 3 1 20619 25 2 Wo YY McCormack AL Shabanowitz J Hunt DF Davis JP Mitchell GL Van Etten RL Sequencing cloning and expression of human red cell type acid phosphatase a
3. transformation in epithelial cells and its oncogenic activities required EphA2 LMW PTP ACP1 negatively regulates EphA2 receptor tyrosine kinase LMW PTP ACP1 is a positive regulator of both tumor onset and development through ephrin EphA2 signaling process and it is a potential target of anticancer drug development Principle of the Assay The Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit is based on an exclusive fluorescence substrate OMFP 3 o0 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 LMW PTP ACP1 y Incubate for 15 min at room temp Add 25 wb of Stop Solution v Measure fluorescence at 510 540 nm emission 482 502 nm excitation Cat CY 1358 2 Version 120420 pry Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit if y gt 4 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 LMW PTP ACPI Assay Buffer 600 uLx1 Below 20 C 10X OMFP 550 uL x1 Below 20 C Recombina
4. 240 uL 480 uL 960 uL 1 440 uL 10X LMW PTP ACP1 AssaysBuffer 5 uL 40 uL 80 uL 160 pL 240 uL 10X OMFP 5 pL 40 uL 80 uL 160 pL 240 uL Total volume of Assay Mixture 40 uL 320 uL 640 uL 1 240 uL 1 920 uL Cat CY 1358 4 Version 120420 Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit 4 gt ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure In order to estimate the inhibitory effect on LMW PTP ACP1 activity by the test compounds correctly it 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 indicated in the Table 1 below When test chemicals cause an inhibitory effect on LMW PTP ACP1 activity the level of increase of fluorescence intensity is weakened as compared with Vehicle control The increase 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 Phosphatase Inhibitor to each well of the microtiter plate and mix well Table 1 Reaction mixture ssavTeacents Test Vehicle Inhibitor No Enzyme y reag Sample j Control Control Control Assay Mixture 40 pL 40 uL 40 uL 40 pL Test Compound Sub Vehicl
5. A Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fluorometric Assay Kit for Measuring LMW PTP ACP1 Phosphatase Activity CycLex Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit 100 Assays Cat CY 1358 Intended US c scscsassessuscscensseecsennseeesnasoecns 1 PMOL ALS suet cucu E EEE T 1 Introduction isch ncaabaesdocsstapcaasistacaadibacess 2 Principle of the Assay cccceseeeeeees 2 Materials Provided 4 vissscsvisnsaveassnccxcounseisess 3 Materials Required but not Provided 3 Precautions and Recommendation 3 4 Detailed Protocol ccccccccccecsesseeeeeseees 4 6 Evaluation of Results ccccceeeeeeees 7 9 Troubleshooting o acsecustcostsonniaccuiinverievsbeds 10 Reagent Stability ssc cssciieeccameinonssannteesntes 10 BR SPSRETIO SS a c0cesxcsreacsesantsecounsdereaterdaveneanss 10 11 Related Proquets i csdvisssanssanedeatuevsacrionatcens 1142 Intended Use The CycLex Research product Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit is a fluorometric and non radioactive assay designed to measure the activity of LMW PTP ACPI1 protein phosphatase This 96 well assay is useful for screening inhibitors and modulators of LMW PTP ACP1 activity in HTS The kit includes all necessary components including recombinant human full length LMW PTP ACP1 for use in preinvestigational drug discovery assay
6. ch 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 1358 12 Version 120420
7. cytoplasmic phosphotyrosyl protein phosphatase JBiol Chem 1992 May 25 267 15 10856 65 3 Chiarugi P Cirri P Raugei Gy Manao G Taddei L Ramponi G Low M r phosphotyrosine protein phosphatase interacts with the PDGF receptor directly via its catalytic site Biochem Biophys Res Commun 1996 Feb 6 219 21 5 4 Chiarugi P Girri P Marra F Raugei G Camici G Manao G Ramponi G LMW PTP is a negative regulator of insulin mediated mitotic and metabolic signalling Biochem Biophys Res Commun 1997 Sep 18 238 2 676 82 5 Fiaschi T Chiarugi P Buricchi F Giannoni E Taddei ML Talini D Cozzi G Zecchi Orlandini S Raugei G Ramponi G Low molecular weight protein tyrosine phosphatase is involved in growth inhibition during cell differentiation J Biol Chem 2001 Dec 28 276 52 49156 63 6 Kikawa KD Vidale DR Van Etten RL Kinch MS Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation J Biol Chem 2002 Oct 18 277 42 39274 9 Cat CY 1358 10 Version 120420 pry Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 7 Chiarugi P Taddei ML Schiavone N Papucci L Giannoni E Fiaschi T Capaccioli S Raugei G Ramponi G LMW PTP is a positive regulator of tumor onset and growth Oncogene 2004May 13 23 22 3905 14 8 Malentacchi F Marzocchini R Gelmini S Orlando C Serio M Rampon
8. e of Test Compounds 5 uL 5 uL 10X Phosphatase Inhibitor 5 uL Recombinant LMW PTP ACP1 20 m units pL SuL 5 uL 5 uL Distilled water 5 uL Total Volume of the Reaction mixture 50 uL 50 uL 50 uL 50 uL 10X Phosphatase Inhibitor ImM Na VOy See Page 4 section Preparation of Reagents 2 Initiate reactions by adding 5 pL of Recombinant LMW PTP ACP1 or distilled 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 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 If neces amp ary 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 1358 5 Version 120420 P Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit ey cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Alternate procedure 1 Following Table 1 above first add Assay mixture to microtiter plate wells Second add Test Compound or Vehicle o
9. f Test Compounds or 10X Phosphatase Inhibitor to each well of the microtiter plate and mix well 2 Initiate reactions by adding 5 uL of Recombinant LMW PTP ACPI1 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 aicrotiter plate fluorometer with excitation at 482 502 nm and emission at 510 540 nm 4 Measure and calculate the rate of reaction while the reaction velocity remains constant Caution and Significance e All samples and Recombinant LMW PTP ACP1 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 1358 6 Version 120420 pry Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit if y 3X User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Analysis of Inhibitor Effect 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 TestsSamples and Vehicle Control 3 Calculate the Intensity Fluorescence Intensity of Test Sample Intensity X 100 Fluorescence Inte
10. i G Raugei G Up regul ted expression of low molecular weight protein tyrosine phosphatases in different human cancers Biochem Biophys Res Commun 2005 Sep 2 334 3 875 83 9 Parri M Buricchi F Taddei ML Giannoni E Raugei G Ramponi G Chiarugi P EphrinA1 repulsive response is regulated by an EphA2 tyrosine phosphatase J Biol Chem 2005 Oct 7 280 40 34008 18 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 FluorometricAssay 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 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 Protei
11. n Tyrosine Phosphatase PTPRK Ist 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 PP5 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 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 E1373 Cat CY 1358 11 Version 120420 pry Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 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 CireuLex products are supplied for resear
12. nsity of Vehicle Control Note This Intensity is a rough value of enzyme activity or inhibition For greater accuracy plot a y 8 y y gr y standard curve of LMW PTP ACP1 for each new set of reactions and estimate the Activity see below Fig l LMW PTP ACP1 Inhibition Curve by Na3VO SQV Sodium Orthovanadate o ja 5 c e O ra e a S gt B ao C oO y oO gt D o oO x 0 0 00001 0 0001 0 001 0 01 0 1 Na3VO Conc mM Cat CY 1358 T Version 120420 Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit Yelex User s Manual For Research Use Only Not for use in diagnostic procedures Analysis of Enzyme Activity LMW PTP ACPI Standard Curve and Activity O 25 Dilute the 10X LMW PTP ACPI Assay Buffer 1 10 with distilled water to make 1X Assay Make serial dilutions of Recombinant LMW PTP ACP1 with 1X Assay Buffer ex 100 6 25 1 56 and 0 N 3 Run reactions with Vehicle and serial dilutions of Recombinant LMW PTP ACP1 as in the Detailed Protocol 4 Plot standard curve data dose dependent curve data as fluorescence intensity a nm versus dose of LMW PTP ACPI 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 LMW P activity for the experimental data Fig 2 Dose Dependency of Recombinant LMW PTP ACP1 Q 2 500 000 2 000 000
13. nt LMW PTP ACP1 20 units uL 500 nL x1 70 C 100X Phosphatase Inhibitor 10 mM Na3VO in DW 100 uL x4 Below 20 C Stop Solution 1 300 uL x2 Below 20 C lInstruction Manual 1 Room temp Recombinant LMW PTP ACP1 is human full length with N terminal GST Tag The GenBank Accession No is BC106011 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 detecttOMFP Pipettors 2 20 uL 20 200 uL and 200 1000 uE 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 sample preparation e Reagent reservoirs Ice bucket to keep reagents cold until use Precautions and Recommendations e Upon receipt store the kit at 70 C Do not exposedeagents to excessive light Do not use kit components beyond the indicated kit expiration date e Rinse all detergent residue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits e Do not mouth pipette
14. or ingest any of the reagents Cat CY 1358 3 Version 120420 Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit User s Manual wy yclex e Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled For Research Use Only Not for use in diagnostic procedures 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 LMW PTP ACP1 and keep all reagents including Recombinant LMW PTP ACP1 on ice untilpuse AVOID REPEATED FREEZE THAW CYCLES OF Recombinant LMW PTP AGPI Making aliquot of Recombinant LMW PTP ACP1 is recommended Use them only after they are completely thawed and mixed 1 Prepare 10X Phosphatase Inhibitor by adding 5 uL of the 100X Phosphatase Inhibitor provided to 45 uL of distilled deionized water Mixqwell Discard any unused 10X Phosphatase Inhibitor after use 2 Prepare Assay Mixture by adding 5 uL of the 10X Assay Buffer provided and 5 uL of the 10X OMFP provided to 30 uL of distilled deionized water per one assay Mix well Assay Mixture Assay reagents lassay 8 assays 16 assays 32 assays 48 assays Distilled water 30 uL
15. s This assay kit is for research use only and not for use in human diagnostic or therapeutic procedures Storage e Upon receipt store the kit at 70 C e Don t expose reagents to excessive light AVOID REPEATED FREEZE THAW CYCLES OF 3 Recombinant LMW PTP ACP1 Cat CY 1358 1 Version 120420 Protein Phosphatase LMW PTP ACP1 Fluorometric Assay Kit yy ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Protein tyrosine phosphorylation plays an essential role in the regulation of many cellular processes including cellular proliferation differentiation migration and tumorigenic transformation The phosphorylation of proteins on tyrosine is catalyzed by numerous protein tyrosine kinases and is rapidly and reversibly dephosphorylated by Protein tyrosine phosphatases PTPases The low molecular weight protein tyrosine phosphatase LMW PTP is an 18 kDa cytos6lie enzyme also known as acidic protein phosphatase 1 ACP1 LMW PTP ACP1 is specific for phosphotyrosine in peptides and proteins but the enzyme shares very limited sequence homology with other PTPases Although LMW PTP ACP1 has been showed as negative regulator of insulin and platelet derived growth factor PDGF mediated mitotic and metabolic signaling LMW PTP ACPT is frequently overexpressed in transformed cell Recent studies suggested that entopic overexpression of LMW PTP ACP1 is sufficient to confer
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