CY-1250 Cathepsin S Fluorometric Assay Kit
Contents
1. 1 Following Table 1 below first add Assay mixture to microtiter plate wells Second add Test Compound or Vehicle of Test Compound or 10X Cathepsin S Inhibitor and 1X Recombinant Cathepsin S to each well of the microtiter plate and mix well ge Optional For best accuracy it is advisable to pre incubate the plate for 5 10 min at assay temperature Table 1 Reaction mixture Assay reacenits Test Vehicle Inhibitor No Enzyme y reag Sample b Control Control Control Assay Buffer 35 uL 35 uL 35 uL 35 uL Test Compound 5 uL Vehicle of Test Compound 5 uL 5 uL 10X Cysteine protease Inhibitor 10 uM E 64 5 uL 1X Recombinant Cathepsin S 5 ng uL SuL 5 uL SuL Assay Buffer 5 uL Total Volume 45 uL 45 uL 45 uL 45 uL See page 4 section Preparation of Reagents within the kit 2 Initiate reactions by adding 5 bof 10X Fluoro Substrate to each well and mixing thoroughly 3 Read fluorescence intensity for 20 30 min or desired length of time at 2 to 5 minute intervals using microtiter plate fluorometer with excitation at 355 380 nm and emission at 440 460 nm at 30 C Any assay temperature from room temperature to 37 C may be used 4 Measure and calculate the rate of reaction while the reaction velocity remains constant Caution and Significance e All samples and Recombinant Cathepsin S should be assayed in duplicate e Use2. amp Lovo No treatment 4 Lovo 37 C pretreatment A431 No treatment A431 37C pretreatment g gt gt wt sa a N N Lag es N z Q 30 40 Time min Cat CY 1250 12 Version 120420 pry Cathepsin S Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The Recombinant Cathepsin S 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 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 were followed accurately such results indicate a need for multi channel pipette maintenance Reagent Stability All of the reagents included in the CycLex Cathepsin S Fluorometric Assay Kit have been tested for stability Reagents should not be used beyond the stated expiratio
3. intensity at 460 nm versus reaction time 4 Determine the reaction time range in which the increase in fluorescence intensity at 460mm 1s linear 5 Calculate activity Fluorescence Intensity of Test Sample Activity reaction velocity Reaction time min NOTE Usually the linear range is from 0 to 15 min This value is variable depending on reaction conditions and storage handling of the Recombinant Cathepsin S Decreasing the amount of Recombinant Cathepsin S in the assay may help to lengthen the time range Fig 1 Typical Time Course Curve Cathepsin S Time Course 125 100 J4 n N n RFU F355 F460 x 10 3 U gt 0 10 20 30 40 50 60 70 Time min Cat CY 1250 8 Version 120420 pry Cathepsin S Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 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 Test Samples and Vehicle Control 3 Calculate the Intensity Reaction velocity of Test Sample Intensity X 100 Reaction velocity of Vehicle Control NOTE This Intensity is a rough value of enzyme activity or inhibition Bor greater accuracy plot a standard curve of Cathepsin S for each new set of reactions afd
4. of a microtiter plate shaker is recommended for complete mixing e If the test compounds or samples themselves emit fluorescence at excitation wavelength 355 380 nm and fluorescence wavelength 440 460 nm the test assay cannot be evaluated correctly Cat CY 1250 Version 120420 pry Cathepsin S Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Cathepsin S Activity Assay Procedure In order to correctly measure the activity of Cathepsin S in cell lysate it is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and Positive control at least once for the first experiment in addition to No enzyme control as indicated in the following table Here Cathepsin S activity is defined as neutral pH treatment resistant cysteine protease activity in cell lysate using this kit Thus the neutral pH treatment of cell lysate is required for measuring Cathepsin S activity in cell lysate See page 11 section Sample Preparation Although the level of fluorescent intensity increases in Test sample when CathepsinyS enzyme activity is in the sample the significant reduced reaction velocity is observed in Inhibitor control and No enzyme control 1 Following Table 1 below first add Assay Buffer to microtiter plate wells Second add 10X Cysteine protease Inhibitor or Vehicle
5. this point the cell pellet can be frozen at 80 C and lyse at a later date 4 Lyse the cell pellet in 0 2 mL of ice cold Cell Lysis Buffer for 30 minutes on ice with vortexing at 10 minute intervals Cell Lysis Buffer 1X 10 mM HEPS pH 7 5 100 mM NaCl 2 mM EDTA 2 mM EGTA 0 2 Triton X 100 2 mM DTT 5 Microcentrifuge at top speed for 10 minutes at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate If necessary cell lysate can be stored at 80 C 6 Before the assay preincubated the cell lysate in Cell Lysis Buffer pH7 5 at 37 C for 60 min in order to inactivate cathepsins Land B which would also hydrolyze same substrate 7 Go to section Cathepsin S Activity Assay Procedure page 6 NOTE THE ABOVE 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 NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED Cat CY 1250 11 Version 120420 pry Cathepsin S Fluorometric Assay Kit f User cA ycLex ser s Manual For Research Use Only Not for use in diagnostic procedures Fig 4 Effect of 37 C pretreatment on cathepsin like protease activities Cathepsin like protease Activities HepG2 No treatment HepG2 37 C pretreatment Sw480 No treatment O Sw480 37 C pretreatment
6. 20 C 5X Recombinant Cathepsin S Human c a 25 ng L 110 uL x4 70 C 100X Cysteine protease Inhibitor 0 1 mM E 64 in DMSO 20 pix 20 C 100X AMC standard 100 uM 7 amino 4 methylcoumarin 50 nL xt 20 C Instruction Manual I Room temp Materials Required but not Provided e Microtiter plate suitable for use with a fluorometric plate reader black microplates provide better signal to noise ratio e Microplate reading fluorometer capable of excitation at a wavelength in the range 355 380 nm and detection of emitted light in the range 440 460 nm 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 sample preparation e Reagent reservoirs e Ice bucket to keep reagents cold until use e DMSO Dimethyl Sulfoxide Sigmas Cat D2438 Precautions and Recommendations Please avoid repeated freezingand thawing of the Recombinant Cathepsin S in this kit There is a possibility that the enzyme activity may be inactivated Aliquot to 10 20 uL and store at 70 C Do not use kit components beyond the indicated kit expiration date Rinse all detergent residue from glassware Use deionized water oftheshighest quality ddH20 Do not mix reagents from different kits Do not mouth pipette or ingest any of the reagents Do not smoke eat or dri
7. 30 40 50 60 70 Time min m Wn oy x H 10 15 Cathepsin S ng Cat CY 1250 10 Version 120420 pry Cathepsin S Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation The following protocols have been shown to work with a number of different cells and enzyme sources and are provided as examples of suitable methods Crude samples can frequently be used without dilution while more concentrated or highly purified cathepsin S should be diluted It is strongly advised that the user always perform an initial experiment to determine the proper dilution to be used in subsequent experiments This need not be any more than a single time point assay using serial dilutions of the crude extract cell lysate or sample fraction taken prior to a purification step All sample preparation should be performed at 4 C and recoveredsfractions should be kept at 70 C to prevent loss of enzymatic activity Preparation of Cell Lysate Note This protocol has been successfully applied to several cell lines Users should optimize the cell extraction procedure for their own applications pa Collect cells in PBS by centrifugation 10 non adherent cells 6r scraping from 10 cm culture dish adherent cells 2 Wash cells twice with cold PBS U Remove and discard the supernatant completely and collect the cell pellet At
8. acture 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 1250 14 Version 120420
9. estimate the Activity see below Fig 2 Typical Inhibition Curve by E 64 Inhibition of Cathepsin S by E 64 5 As z 0 1E 05 0 0001 0 001 0 01 0 1 E64 conc uM Cat CY 1250 9 Version 120420 4 Cathepsin S Fluorometric Assay Kit 4 j c ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Analysis of Inhibitor Effect 2 Cathepsin S Standard Curve and Activity Dilute the 8X Assay Buffer 1 8 with distilled water to make 1X Assay Buffer 2 Make serial dilutions of Recombinant Cathepsin S with 1X Assay Buffer ex 100 50 25 12 5 6 25 3 13 and 0 Run reactions with Vehicle and serial dilutions of Recombinant Cathepsin S as described in the Detailed Protocol 4 Plot standard curve data as fluorescence intensity at 460 nm versus dose of Cathepsin S ng assay 5 Obtain a line fit to the data using appropriate calculations Use the slope and Y intercept to calculate the amount of Cathepsin S activitysfor fhe experimental data U an Fig 3 Typical Dose Dependency Curve jee Cathepsin S Dose Dependency e 25 ng e 12 5 ng 100 M i e 6 25ing pot er a ages T 3 125 ng 1 5625 a 75 bes ng be ited Oo tes Se a o o S He es So OH 6 eH 0 MA RQ E eee ree a ag a EEE T E os A SO pae amp er eee a ee ee i SSS EEE 0 10 20
10. l For Research Use Only Not for use in diagnostic procedures Determine microplate reader conversion factor for AMC fluorophore The exact AMC concentration range that will be useful for preparing a standard curve will vary depending on the fluorometer model the gain setting and the exact excitation and emission wavelengths used Please dilute the 100X AMC standard provided 100 uM to 1 0 uM as the highest standard and make 4 fold serial dilution with Assay Buffer and then measuring the fluorescence of 50 ul in a microtiter plate fluorometer with excitation at 355 380 nm and emission at 440 460 nm Th stimate of uM RFU obtained with this measurement together with the observed range of values obtained in the enzyme assays can then be used to plan an appropriate series of dilutions for a standard curve dhe slope of the standard curve can then be used as the uM RFU conversion factor Typiacl AMC Standard Curve AMC Standard Curve 75 000 vt ey N N Lag e2 kez z y 69296x 89 725 R 1 0 5 0 8 AMC conc uM Cat CY 1250 7 Version 120420 pry Cathepsin S Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results 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
11. my Cathepsin S Fluorometric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fluorometric Assay Kit for measuring Cathepsin S Activity CycLex Cathepsin S Fluorometric Assay Kit P For 100 Assays Intended USE a pe puscen cca tanaetseesnaeutesangeeneennecnnts 1 USES east ean E 1 PAGO GU CLIN 5 oc ssacssaduisesecesseestarspandessavasssoesens 2 Principle OF the Assay sscvsssissecsuvvdasoasswavsoavns 2 Materials Provided cccccccssssececessseeees 3 Materials Required but not Provided 3 Precautions and Recommendation 3 Detailed Protocols iictisscssssonsistascesmeccsebss 4 7 Evaluation of Results ccccccceceesseeeeees 8 10 Sample PreparatiOn xgscecaiassenicudsdyaiatarenincteans 11 Troubleshooting is scssssssccsssssscuvsassnccosssaccnass 12 Reagent SADT ITY wis scuaswrstataemmneavananncsesvens 12 RGIPENCES erisassscsaeidin anna 12 Related Products cccccesscceceesseeceessseees 13 Intended Use The CycLex Research Product CycLex Cathepsin S Fluorometric Assay Kit detects cathepsin S protease activity in cell lysates Primarily the CycLex Research Product CycLex Cathepsin S Fluorometric Assay Kit is designed for the rapid and sensitive evaluation of cathepsin S inhibitors using recombinant cathepsin S Additionally many cultured primary cells or cell lines can be assayed for cathepsin S protease activity with the CycLex Research Product C
12. n date Upon receipt all kit reagents except Recombinant Cathepsin S should be stored at 20 C Recombinant Cathepsin S should be stored at 70 C After use return kit reagents to 20 C as soon aspossible For research use only not for use in human diagnostic or therapeutic procedures References 1 Honey K and AY Rudensky Nat Rev Immunol 3 472 2003 2 Hsieh CS et al J Immunol 168 2618 2002 3 S N Desai et al Eur J Pharmacol 538 468 2006 4 J O Link and S Zipfel Curr Opin Drug Discov Devel 9 471 2006 5 Kenneth J et al Arterioscler ThrombyVasce Biol 26 851 2006 6 Aikawa E et al Circulation Aprih7 2009 119 13 1785 7 de Nooijer R et al Arterioscler Thromb Vasc Biol 29 188 2009 8 Wang B et al J Biol Chem4281 6020 2006 9 Flannery T et al Int J Cancers119 854 2006 10 Cynthia A et al Am J Patholed 46 848 1995 Cat CY 1250 13 Version 120420 pry Cathepsin S Fluorometric Assay Kit rai S y gt X User s Manual For Research Use Only Not for use in diagnostic procedures Related Products Cysteine Protease Cathepsin S Cat CY E1250 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 manuf
13. nk when performing the assay or in areas where samples or reagents are handled Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly Cat CY 1250 3 Version 120420 pry Cathepsin S Fluorometric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CycLex Cathepsin S Fluorometric Assay Kit is provided with concentrated reagents Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Reagents within the kit Thaw the reagents at room temperature except Recombinant Cathepsin S and keepyall reagents on ice until use Use them only after they are completely thawed and mixed 1 Prepare Assay Buffer by adding 5 uL of the 8X Assay Buffer provid d to 35 uL of distilled deionized water per one assay Mix well Unused buffer should be stored at 20 C Discard any unused Assay Buffer after use Assay Buffer Assay reagents l assay 8assays A6 assays 32 assays 48 assays Distilled water 35 uL 280uL 560 uL 1 120uL 1 680 nL 8X Assay Buffer SuL 40 uL 80 uL 160 uL 240 uL Total volume of Assay Buffer 40 uL 320 pL 640 uL 1 280 uL 1 920 uL 2 Prepare 1X Recombinant Cathe
14. of Cysteine protease Inhibitor and 1X Recombinant Cathepsin S or Your enzyme fraction Neutral pH treated or Buffer for cell lysate to each well of the microtiter plate and mix well Optional For best accuracy it is advisable to pre incubate the plateyfor 5 10 min at assay temperature Table 2 Reaction mixture Assay reagents Test Sample Inhibitor Positive No enzyme control control control Assay Buffer 35 pL 35 pL 35 pL 35 pL 10X Cysteine protease Inhibitor 10 uM Vehicle of 10X Cysteine protease Inhibitor 1X Recombinant Cathepsin S 5 ng uL Buffer for cell lysate Total Volume 45 pL 45 pL 45 pL 45 pL See page 4 section Preparation of Reagents within the kit See page 11 section Sample Preparation 2 Following the above table add the Reagents to each well of the microplate Finally initiate the reaction by adding 5 uL of 10X Fluoro Substrate to each well and mixing thoroughly 3 Read fluorescence intensity for 20 30 min or desired length of time at 2 to 5 minute intervals using microtiter plate fluorometer with excitation at 355 380 nm and emission at 440 460 nm at 30 C Any assay temperature from room temperature to 37 C may be used 4 Measure and calculate the rate of reaction while the reaction velocity remains constant Cat CY 1250 6 Version 120420 4 Cathepsin S Fluorometric Assay Kit 4 j c ycLex User s Manua
15. psin S by diluting the 5X Recombinant Cathepsin S provided 1 5 in 1X Assay Buffer Mix well Put it in ice Discard any unused 1X Recombinant Cathepsin S after use Assay reagents lassay 8 assays 16 assays 32 assays 48 assays 1X Assay Buffer 8 uL 40 uL 72 uL 136 uL 200 uL 5X Recombinant Cathepsin S 2 uL 10 uL 18 uL 34 uL 50 uL Total volume 10 uL 50 uL 90 uL 170 uL 250 uL 3 Prepare 10X Cysteine protease Inhibitor 10 uM E 64 by adding 2 uL of the 100X Cysteine protease Inhibitor provided to 18 uL of distilled deionized water Mix well Discard any unused 10X Cysteine protease Inhibitor after use Cat CY 1250 Version 120420 yy wyclex Inhibitor Screening Assay Procedure User s Manual For Research Use Only Not for use in diagnostic procedures Cathepsin S Fluorometric Assay Kit In order to estimate the inhibitory effect on Cathepsin S activity by the test compounds corfectly 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 Cathepsin S 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
16. uchsas rheumatoid arthritis multiple sclerosis 3 4 atherosclerosis 5 7 cancer 8 9 Alzheimer s and Down s Syndrome 10 It is an attractive target for drug discovery due to its involvement in immune system function as well as extracellular matrix breakdown Principle of the Assay The CycLex Cathepsin S Fluorometric Assay Kit is based onan exclusive fluorescence substrate Z Val Val Arg MCA Benzyloxycarbonyl L Valyl L Valyl L Arginine 4 Methyl Coumaryl 7 Amide 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 containing Recombinant Cathepsin S and 5 uL of test compound in the wells v Add 5 uL of 10X Fluoro Substrate Y Measure velocity of fluorescence intensity with excitation at 355 380 nm and emission at 440 460 nm for 20 30 gt min at 30 C Cat CY 1250 2 Version 120420 pry Cathepsin S Fluorometric Assay Kit if y 3X User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and Recombinant Cathepsin S should be assayed in duplicate The following components are supplied and are sufficient for one hundred assays Components of Kit Components Quantity Storage 8X Assay Buffer 800 nLx1 20 C 10X Fluoro Substrate 100 uM Z Val Val Arg MCA 550 pL x1
17. ycLex Cathepsin S Fluorometric Assay Kit if the appropriate treatment of cathepsin S is conducted Applications for this kit include 1 Monitoring cathepsin_S activity at purification step 2 Screening inhibitors or activators of cathepsin S 3 Evaluating the effects of pharmacological agents on cathepsin S activity This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store Recombinant Cathepsin S at 70 C and all other components below 20 C e Don texpose reagents to excessive light Cat CY 1250 1 Version 120420 Cathepsin S Fluorometric Assay Kit yy ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Cathepsin S is a cysteine protease found in the lysosome of lymphatic cells It is a member of the papain superfamily and has 57 identity to cathepsins L and K Unlike most other lysosomal proteases that are only active under acidic conditions the activity of cathepsin S exhibits a broad pH optimum that extends to alkaline pH Cathepsin S is expressed mainly in dendritic cells B cells and macrophages where cathepsin S acts as a key enzyme in major histocompatibility complex class H mediated antigen presentation 1 2 Cathepsin S is upregulated by IFNy through the IFN stimulated response element in the cathepsin S promoter This enzyme is also implicated in the pathogenesis of several diseases S
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