or BTK [C481S] (cat. # CK-01
Contents
1. eicen Assay Innovations ClariCELL Kinase Assay Kit for BTK cat CK 01 1001 or BTK C481S cat CK 01 1002 User s Manual V1 4 20130918 For Research Use Only Not for use in diagnostic procedures or in humans Information in this document is subject to change without notice IMPORTANT SAFETY INFORMATION This assay kit is designed for use by trained scientific professionals following appropriate laboratory safety procedures Appendix A outlines important general safety precautions for utilizing these materials NOTICE TO PURCHASER LIMITED LICENSE BY OPENING PACKAGING CONTAINING THIS PRODUCT RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THE LIMITED USE LICENSE AGREEMENT The full terms of the Limited Use License Agreement can be found in Appendix B of this document or by contacting Cell Assay Innovations Inc at 100 Cummings Center Suite 424J Beverly MA 01915 Product Numbers CK 01 1001 and CK 01 1002 1 www cellassayinnov com eicen Assay Innovations Table of Contents Ti FOGG OM ess sssrini saa ret iiae e i E a E e ei E ae 3 Overview of Experimental Procedure iiiscc lt sccenseascccaseneassensSeseseatonstasspeoasnnecesasees 4 Materials and Storage Conditions sss susrete rirerire serrr ssrrrsrsrrrrrsrrerrrsrrrrn 4 5 Assay F ROU CO ereet E rE SE TEE EE EEE 5 8 Example Plate Layout and Expected Results 0 0 ccc cccccecceeteee eee sees cesses ee 9 10 BR eG 6 cee acetate ceca cals oa A AE2. gross negligence or willful misconduct of CAI 8 Disclaimer of Warranties CAI warrants that the accompanying datasheet correctly describes the activity of the Materials and their suitability for performing assays described in the protocol CAI disclaims any other representations and warranties expressed or implied including without limitation any warranty of noninfringment title merchantability or fitness for a particular purpose 9 Limitation of Liability To the fullest extent allowed by law in no event shall CAI be liable whether in contract tort warranty or under any statute or on any other basis for special incidental indirect punitive multiple or consequential damages in connection with or arising from this document agreement or license including but not limited to the use thereof whether or not foreseeable and whether or not CAI is advised of the possibility of such damages 10 Entire Agreement and Assignability This Limited Use Agreement sets forth the complete and entire agreement of the parties with respect to the subject matter hereof and supersedes and terminates all prior agreements and understandings between the parties No subsequent amendment or addition to this Limited Use Agreement shall be binding upon the parties unless reduced to writing and signed by the respective authorized officers of the parties This Limited Use Agreement shall not be assigned or otherwise transferred by Recipient 11 Governing La
3. placing it in a 37 C water bath 2 min Be careful to not incubate the cells longer than is necessary to thaw the cells as the viability may be impacted Transfer the cells to a 15 mL centrifuge tube and add 5 mL of HEK293 culture medium Rinse the cryotube with an additional 1 mL of medium then combine the rinse with the solution in the 15 mL tube Spin the cells for 6 minutes at 140 g approximately 900 rpm Carefully remove and discard the supernatant without disturbing the cell pellet Resuspend the cells in 500 uL of HEK293 culture medium Pipet up and down several times with a 1 mL pipettor and tip to break up cell clumps Optional Count the cell number and determine the cell viability using standard techniques such as trypan blue staining An optimal dilution for counting using a hemocytometer is 1 4 dilution of cells followed by 1 2 with trypan blue For example use 10 uL of cells plus 30 uL of medium plus 40 uL of trypan blue for counting Note The cell viability should be gt 70 and the total cell number should be 1 5 x 10 cells per vial for BTK and 1 3 x 10 cells per vial for BTK C4818 Dilute the cells with HEK293 culture medium For wild type BTK add 5 5 mL of culture medium for a total volume of 6 mL and for BTK C481S cells add 5 mL of culture medium for a total volume of 5 5 mL Pipet up and down to ensure that the cells are evenly distributed Dispense 45 uL of the cells per well to a 96 well
4. 293 cells that have already been transfected with a vector encoding sequence verified full length human BTK or BTK C481S plus reagents suitable for ELISA detection of BTK Product Numbers CK 01 1001 and CK 01 1002 3 www cellassayinnov com vice Assay Innovations or BTK C481S autophosphorylation levels The reagents have been characterized extensively and titrated appropriately such that the kit can easily be utilized without further optimization for testing the ability of small molecules to modulate the kinase activity of BTK or BTK C4818 in human cells in a high throughput manner Overview of Experimental Procedure e Thaw and wash ClariCELL cells e Dispense cells into 96 well plates YOELI e Incubate for 1 hour at 37 C humidified with 5 CO e Add test compounds to cells in 96 well plate WAG e Incubate for 2 hours at 37 C humidified with 5 CO Compound e Lyse cells and transfer lysate to ELISA plate e Perform ELISA with phospho specific antibody Detect Kinase E Quantify substrate phosporylation levels e Compare phosphorylation levels of compound treated Analyze samples vs controls Results Materials Supplied and Storage Conditions e 1 vial cryotube of ClariCELL BTK cells 1 5 x 10 cells in 500 uL volume or BTK C481S cells 1 3 x 10 cells in 500 uL volume per assay plate to be stored at 80 C for short term 3 months or less For longer term store in the vapor phase of liquid ni
5. Recipient additionally agrees to not reverse engineer or utilize any component s of this product and or its documentation to establish the assay system represented by the product internally or elsewhere independently of CAI Recipient has no right to propagate modify derivatize genetically engineer or otherwise create variations of the cells contained in this assay kit For information on obtaining additional rights contact CAI directly at 100 Cummings Center Suite 424J Beverly MA 01915 Product Numbers CK 01 1001 and CK 01 1002 11 www cellassayinnov com eicen Assay Innovations 6 Compliance with Laws Precautions When utilizing the Materials Recipient shall use its expertise and facilities in strict accordance with all applicable local state and federal laws regulations and guidelines Recipient understands that the Materials may have biological and or chemical properties that are unpredictable and unknown at the time of transfer that they are to be used with caution and prudence and that they will not to be used for testing in or treatment of humans 7 Liability Recipient assumes all liability for damages that may arise from the use storage or disposal of the Materials CAI will not be liable to Recipient for any loss claim or demand made by the Recipient or made against the Recipient by any other party due to or arising from the use of the Materials by the Recipient except to the extent permitted by law when caused by the
6. TS E AEAEE EAEE eE a EAE 10 Appendix A Important Safety Information 0 c ec ence eee eee 11 Appendix B Limited Use License Agreement ceeeceececeee ence eeceeeeecnaee 11 12 Product Numbers CK 01 1001 and CK 01 1002 2 www cellassayinnov com cicen Assay Innovations Introduction BTK Bruton s agammaglobulinemia Tyrosine Kinase is a member of the Tec family of cytoplasmic tyrosine kinases BTK is an important regulator of B cell development and signaling 1 and as such has been investigated as a target of inhibition for the treatment of B cell malignancies autoimmune disorders and inflammation 2 The activity of BTK is regulated by a variety of mechanisms including membrane translocation and phosphorylation Key in the activation process is phosphorylation of two tyrosine residues Y551 and Y223 1 It is generally believed that Y551 of BTK is phosphorylated by a Src family kinase likely Lyn which subsequently leads to autophosphorylation of the Y223 site 3 1 However BTK has also been shown to autophosphorylate at the Y551 site at least in vitro 4 BTK C481S is a variation of wild type BTK where the cysteine at amino acid 481 has been mutated to serine The clinical compound PCI 32765 ibrutinib irreversibly binds BTK by reacting with the cysteine at position 481 5 As such ibrutinib is predicted to exhibit reduced binding and potency toward BTK C481S compared to the wild type protei
7. e g Selleck Chemical cat S2680 e DMSO e TBST e g Fisher Scientific cat PI 28360 for 20x concentrate e 1 Step Slow TMB ELISA e g Pierce cat 34024 e 15 mL amber dark tubes or aluminum foil e 2MH gt 2SO e Optional Trypan Blue Instrumentation and Equipment Required e Centrifuge suitable to spin 15 mL tubes e Absorbance microplate reader 450 nm e Microplate Shaker e 37 C water bath e Cell Culture Incubator humidified at 37 C and 5 CO e Optional Cell Counter Assay Protocol 1 Coat the wells of a 96 well half area ELISA plate with the provided coating antibody red capped tube Dilute the coating antibody to 0 01 mg mL in PBS For each 96 well plate add 50 uL of coating antibody to 5 mL of PBS and add 50 uL solution per well Incubate for 2 hours at room temperature RT or overnight at 4 C 2 Shake off the antibody solution from the 96 well ELISA plate Add 85 uL 1 BSA solution per well for blocking Incubate for 30 minutes at RT with shaking or overnight at 4 C Product Numbers CK 01 1001 and CK 01 1002 5 www cellassayinnov com eicen Assay Innovations Note After shaking off the solution from plates tap the plate on absorbent paper to blot off the residual liquid The same technique can be used for plate washing in subsequent steps 3 Prepare the cell suspension from frozen cells as follows Thaw one cryotube of BTK or BTK C481S ClariCELL frozen cells per assay plate by
8. es of compound concentrations utilizing appropriate curve fitting software e g GraphPad Prism software 18 Fit the IC50 curves utilizing standard techniques e g sigmoidal dose response curve fitting to determine IC50 values Note that total phospho tyrosine is the readout measured in these assays and therefore there is a possibility that some tyrosine kinase activity in addition to that of BTK or BTK C481S can be detected Also note that compounds exhibiting extreme cytotoxicity will appear to be BTK or BTK C481S inhibitors in the assays However since the compound incubation time is relatively short 2 hours this risk is considered to be low If suspected cytotoxicity should be assessed in a separate assay Product Numbers CK 01 1001 and CK 01 1002 8 www cellassayinnov com sicen Assay Innovations Example Plate Layout and Expected Results A dose response curve for ibrutinib was generated utilizing the ClariCELL BTK assay kit 8 doses of ibrutinib were tested starting at 1 uM testing concentration and making 1 to 3 dilutions The assay protocol was followed as outlined above and the plate layout was as shown below 2 1 0 10 10 1 3 1 0 Ei Ging war Absorbance Reading at 450 nm 1 2 3 Se a SG iles ose Plate Statistics average positive control 0 307 average negative control 0 081 signal to background 0 307 0 081 3 8 Z value 0 71 Calculated Percent Inhibi
9. greement the Limited Use Agreement constitutes a legal agreement between the addressee and his or her organization collectively Recipient and Cell Assay Innovations Inc CAT 2 Controlling Terms This Limited Use Agreement sets forth herein the only terms and conditions governing the use of the enclosed product 3 License Grant Subject to the terms and conditions of this Limited Use Agreement CAI hereby grants to Recipient a non transferable non exclusive license to use the enclosed assay kit the Materials for research purposes only 4 Use Only by Recipient Recipient will not transfer the Materials to any person or entity except its employees nor authorize any third party to use or sell Materials any components or derivatives thereof 5 Research Use Only Materials shall be used for Research purposes only the Research As used herein the definition of Research excludes uses of the Materials to i perform contract research i1 produce or manufacture products for general sale or iii conduct research activities that result in any sale lease license or transfer of the Material any components or derivatives thereof The product and or any of its components thereof is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of Recipient s activities for a fee or other form of consideration
10. ms of resistance to BTK inhibitor ibrutinib in chronic lymphocytic leukemia CLL Poster Display and Discussion Session at ASCO Chicago IL May 31 June 4 2013 www cellassayinnov com Zhang JH Chung TDY Oldenburg KR A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays 1999 J Biomol Screen 4 67 73 Product Numbers CK 01 1001 and CK 01 1002 10 www cellassayinnov com cicen Assay Innovations Appendix A Important Safety Information The modified HEK293 cells included in this assay kit are classified as biosafety level 2 BSL 2 Containment waste disposal and laboratory procedures appropriate for BSL 2 should be followed For specific hazards associated with the kit components refer to the MSDSs Material Safety Data Sheets For assistance in general guidelines and US regulations regarding BSL 2 please refer to the CDC Center for Disease Control Publication entitled Biosafety in Microbiological and Biomedical Laboratories As with other laboratory procedures researchers utilizing this kit should wear appropriate PPE Personal Protective Equipment such as gloves safety glasses and a lab coat to minimize the risk of exposure to the components Appendix B Limited Use License Agreement READ CAREFULLY BY OPENING PACKAGING ENCLOSING THE MATERIALS RECIPIENT ACCEPTS THE FOLLOWING TERMS AND CONDITIONS 1 Legal Agreement This Limited Use License A
11. n Consistent with this prediction the BTK C481S mutant has recently been reported in ibrutinib resistant patients 6 ClariCELL kinase assays 7 represent an invaluable system for testing the inhibitory activities of small molecules against a specific kinase of interest in the context of human cells Cell based compound potency measurements are important components of the drug discovery process since biochemical potency values often do not translate to cellular activity for a number of reasons including compound membrane permeability cellular ATP concentration compound localization etc With the ClariCELL BTK and BTK C481S assay kits autophosphorylation of human full length wild type BTK or BTK C481S is quantified and the cellular potencies of compounds that modulate these autophosphorylation events are measured Figure 1 depicts an overview of the ClariCELL assay system Expression Vector Figure 1 Basis of for Enzyme Substrate ClariCELL kinase cell based assay technology HEK293 cells ClariCELL are first transiently transfected of interest with or without a separate protein substrate Following compound treatment ELISA assay and cell lysis phosphorylation of or comparable the substrate is quantified by ELISA using an antibody to the Incubate Transfer phosphorylation site sees Platform SET T with a vector encoding a kinase The ClariCELL BTK and BTK C481S kinase assay kits provide HEK
12. s solution and should be added to the lysis buffer just prior to use Note that a different stock concentration of PMSF can be utilized with appropriate adjustment of the amount added such that the final concentration is 5 mM 7 Add 12 5 uL of the complete 5x lysis buffer directly into each well of the tissue culture plate to lyse the cells No medium removal or washing of the cells is necessary Shake the plate for 10 minutes at RT Note Take care in pipetting the lysis buffer as the solution is viscous and also may form bubbles if air is introduced by pipetting or shaking Addition of the lysis buffer will change the color of the medium from pink to yellow orange 8 Prepare the ELISA plate by shaking off the 1 BSA blocking solution Transfer 50 uL of the cell lysate from the tissue culture plate to each corresponding assay well of the ELISA plate and incubate for 1hr at RT with shaking Note The majority of the sample is transferred to the ELISA plate only 12 5 LL of dead volume To ensure that the full amount can be aspirated from the wells it is useful to tilt the plate and pipet carefully from the bottom edges of the wells 9 Shake off the cell lysate solution from the ELISA plate and wash 3x with TBST Note Utilize 150 uL per well of wash solution to ensure thorough washing Product Numbers CK 01 1001 and CK 01 1002 7 www cellassayinnov com eicen Assay Innovations 10 Dilute the provided detection antibody
13. tion Values 1 2 3 616 486 89 ITOnmIOoOw gt Product Numbers CK 01 1001 and CK 01 1002 www cellassayinnov com cicen Assay Innovations IC50 Curve Generated Using GraphPad Prism Software Ibrutinib Curve IC50 0 012uM INH 10 108 107 108 cmpd M Sigmoidal dose response variable slope Best fit values BOTTOM 2 219 TOP 102 3 LOGEC50 7 913 HILLSLOPE 1 357 EC50 1 223e 008 References 1 Mohamed AJ et al Bruton s tyrosine kinase Btk function regulation and transformation with special emphasis on the PH domain 2009 Immunol Rev 228 58 73 Vargas L Hamasy A Nore BF and Smith CIE Inhibitors of BTK and ITK State of the New Drugs for Cancer Autoimmunity and Inflammatory Diseases 2013 Scand J Immunol 78 2 130 9 Rawlings DJ Scharenberg AM Park H Wahl MI Lin S Kato RM Fluckiger AC Witte ON and Kinet JP Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases 1996 Science 271 822 825 Dinh M Grunberger D Ho H Tsing SY Shaw D Lee S Barnett J Hill RJ Swinney DC and Bradshaw JM Activation Mechanism and Steady State Kinetics of Bruton s Tyrosine Kinase 2007 J Biol Chem 282 8768 8776 Pan Z et al Discovery of Selective Irreversible Inhibitors for Bruton s Tyrosine Kinase 2007 ChemMedChem 2 58 61 Chang B et al Tumor genomic profiling reveals mechanis
14. tissue culture plate Ensure that the cells are evenly distributed during transfer to the plate by pipetting up and down after addition to each row or column Note Variations in cell number from well to well will adversely affect the results in terms of data variability The final cell number in the assay should Product Numbers CK 01 1001 and CK 01 1002 6 www cellassayinnov com eicen Assay Innovations be 7 000 10 000 viable cells per well with a consistent number of cells from well to well e Cover the plate with a lid and incubate in a humidified 5 COz incubator at 37 C for 1 hour before compound addition 4 Prepare inhibitors at 10x final assay concentration in 5 v v DMSO When preparing dilution curves always dilute compounds in 100 DMSO before adding water or medium in the final step 5 Add 5 uL of diluted compound to the cells for a final assay concentration of 1x compound and 0 5 DMSO For positive assay controls full activity add 5 uL of 5 DMSO and for negative controls no activity add 5 uL of 100 uM ibrutinib in 5 DMSO final concentration of ibrutinib will be 10 uM Cover the plate with a lid and incubate in a humidified 5 CO incubator at 37 C for 2 hours 6 Prepare complete 5x lysis buffer by adding 200 mM PMSF to the supplied 5x lysis buffer base green capped tube to a concentration of 5 mM 38 uL of 200 mM PMSF to the supplied 1 46 mL 5x lysis buffer base Note PMSF is unstable in aqueou
15. trogen Be sure to follow relevant safety precautions including the use of appropriate gloves and a face shield when removing vials from liquid nitrogen e 1 96 well half area ELISA plate per assay sealed with a clear plate seal to be stored at room temperature Provided for orders of 5 assays or less For orders of 6 or more assays these can be purchased from Corning Costar cat 3690 e 1 tube of coating antibody red capped tube 50 uL per assay at 1 mg mL to be stored at 20 C e 1 tube of detection antibody yellow capped tube 1 uL per assay at 0 2 mg mL to be stored at 4 C Product Numbers CK 01 1001 and CK 01 1002 www cellassayinnov com eicen Assay Innovations e 1 tube of 5x lysis buffer base per assay green capped tubes 1 46 mL each to be stored at 20 C Materials Required but not Supplied e 15 mL centrifuge tubes e 1 half area 96 well high binding clear ELISA plate per assay For orders of 6 or more assays Corning Costar cat 3690 For orders of 5 or less assays plates are included in the kit e PBS e g Fisher Scientific cat BP2438 20 e 1 BSA solution in PBS e g 1 w v solution of Sigma cat A7888 dissolved in PBS e HEK293 culture medium DMEM NEAA 10 FBS e 1 96 well tissue culture plate per assay sterile with lid e PMSF Phenylmethanesulfonyl Fluoride e g Sigma cat 78830 dissolved in isopropyl alcohol to 200 mM e Control BTK inhibitor ibrutinib
16. w This License Agreement shall be construed and governed in accordance with the laws of the Commonwealth of Massachusetts without regard to its conflicts of laws provisions 12 Publication Any publication or presentation of the results of the research using the Materials will duly acknowledge CAI as their source 13 Patents and Trademarks This product is covered by International Patent Application No PCT US13 48887 No right under any other patent claim such as claims to methods apparatus or reagents is conveyed expressly by implication or by estoppel It is the responsibility of the Recipient to determine whether additional IP rights are required to utilize the Materials The trademarks mentioned herein are the property of CAI 2013 Cell Assay Innovations Inc All rights reserved Product Numbers CK 01 1001 and CK 01 1002 12 www cellassayinnov com
17. yellow capped tube 1 5000 in TBST and add 50 ul well For each 96 well plate add 1 uL of antibody to 5 mL of TBST Incubate for 1 hour at RT with shaking Note The plates can alternatively be incubated with antibody overnight at 4 C 11 Take 5 mL per assay plate of the 1 Step Slow TMB ELISA solution out of the 4 C approximately 1 hour before the detection stage step 13 to allow it to equilibrate to RT To protect the 1 Step Slow TMB ELISA from excess light utilize amber dark tubes or wrap the tubes in aluminum foil 12 Shake off the detection antibody solution from the ELISA plate and wash 3x with TBST 13 Add 50 uL per well of TMB ELISA solution and shake for 10 to 15 minutes to allow the blue color to develop Stop the reaction by adding 50 uL of 2M H2501 The blue color should change to yellow 14 Measure the absorbance of the wells at 450 nm 15 Calculate inhibition values from the absorbance readings based on positive and negative control values and according to the following formula INH positive control sample positive control negative control 100 16 If desired calculate Z values based on the following formula 1 3 standard deviation of positives 3 standard deviation of negatives average positive average negative 8 A Z value of greater than or equal to 0 4 generally indicates an acceptable value 17 For dose response curves plot inhibition values vs the log valu
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