Contact Information 70-270 DRx_UM_PHeX_RTK_0514V6 DiscoveRx Corporation (World Wide Headquarters) 42501 Albrae Street ® Fremont, CA 94538 PathHunter eXpress Receptor United States Tyrosine Kinase Functional t | 510.979.1415 f | 510.979.1650 Assay Kit toll-free | 866.448.4864 DiscoveRx Corporation Ltd. (Europe Headquarters) Faraday Wharf, Holt Street User Manual Birmingham Science Park Aston Birmingham, B7 4BB United Kingdom t | +44.121.260.6142 f | +44.121.260.6143 KINOMEscan® A division of DiscoveRx 11180 Roselle Street, Suite D San Diego, CA 92121 United States t | 800.644.5687 f | 858.630.4600 BioSeek® A division of DiscoveRx 310 Utah Avenue, Suite 100 South San Francisco, CA 94080 United States t | 1.650.416.7600 f | 1.650.416.7625 www.discoverx.com © 2014 DiscoveRx Corporation, Fremont, CA 94538. All rights reserved. Simple Solutions for Complex Biology CONTENTS Appendix Target Partner Cell Line eXpress Reference DiscoveRx Cell Plating LEGAL SECTION PAGE 3 Protein Product Product Code Agonist Part # Reagent Code DiscoveRx Part # INTENDED USE PAGE 4 Receptor Tyrosine Kinase Assays TECHNOLOGY PRINCIPLE PAGE 4 C-MET GrB2 93-0632C3 HGF 92-1140 93-0563R16A DDR1 SHC1 93-0578C3 93-0578E3CP16 Collagen Type II 92-1090 93-0563R16A STORAGE CONDITIONS PAGE 5 DDR2 PLCG1 93-0711C3 Collagen Type II 92-1090 93-0563R16A MATERIALS PROVIDED PAGE 5 EphB4 SHC1 93-0468C3 93-0468E3CP16 Ephrin B2 FC 92-1089 93-0563R16A MATERIALS NOT PROVIDED PAGE 6 ErbB1 PLCG1 93-0681C3 EGF 92-1113 93-0563R16A ErbB2/ GrB2 93-0535C3 93-0535E3CP16 Heregulin 92-1031 93-0563R16A MEDIA REQUIREMENTS PAGE 6 ErbB3 ErbB4 SHC1 93-0465C3 NRG1 92-1091 93-0563R16A ASSAY PROCEDURE - AGONIST DOSE RESPONSE PAGE 6 FGFR4 PLCG2 93-0467C3 93-0467C3CP16 FGF-Basic 92-1027 93-0563R16A QUICK START PROCEDURE - AGONIST DOSE RESPONSE PAGE 9 Flt3 P85 93-0506C3 FLT3-Ligand 92-1029 93-0563R16A ASSAY PROCEDURE - ANTAGONIST DOSE RESPONSE PAGE 10 IGFR1 SHC1 93-0505C1 93-0505E1CP17 IGF1 92-1030 93-0563R17A INSR PLCG1 93-0466C3 93-0466E3CP16 Insulin 92-1088 93-0563R16A QUICK-START PROCEDURE - ANTIBODY DOSE RESPONSE PAGE 12 PDGFRb PLCG1 93-0469C3 93-0493E3CP16 PDGFA-B 92-1028 93-0563R16A FREQUENTLY ASK QUESTIONS PAGE 13 TrkA SHC1 93-0462C3 93-0462E3CP16 b-NGF 92-1023 93-0563R16A PLATE MAPS PAGE 14 TrkA- SHC1 93-0529C3 93-0529E3CP16 b-NGF 92-1023 93-0563R16A P75 TrkB SHC1 93-0463C3 BDNF 92-1024 93-0563R16A TrkB- SHC1 93-0530C3 93-0530E3CP16 BDNF 92-1024 93-0563R16A P75 ATTENTION: TrkC SHC1 93-0464C3 93-0464E3CP16 NT3 92-1025 93-0563R16A Read the entire product insert prior to beginning the assay. Refer to TrkC- SHC1 93-0531C3 NT3 92-1025 93-0563R16A the data sheets for additional information on cell-line specific media P75 requirements. Cytosolic Tyrosine Kinase Assays PRLR- PLCG2 93-0686C3 Prolactin 92-1144 93-0563R0A For additional information or Technical Support, contact DiscoveRx or JAK1 visit www.discoverx.com. PRLR- PLCG2 93-0687C3 Prolactin 92-1144 93-0563R0A JAK2 2 15 A. Agonist Dose Response (96-well plate layout): LEGAL SECTION This product and/or its use is covered by one or more US and/or foreign patents, patents applications, and trade secrets that are either owned or licensed to DiscoveRx corporation. For some products/cell lines, certain third party gene- specific patents may be required to use the cell line. It is Purchaser’s responsibil- ity to determine if such patents or other intellectual property rights are required. DiscoveRx® and PathHunter® are registered trademarks of DiscoveRx Corporation. DiscoveRx name and logo are a trademark of DiscoveRx Corporation. GraphPad Prism is a registered trademark of GraphPad Software, Inc. © 2014 DiscoveRx Corporation. All rights reserved. All goods and services sold are subject to the terms and conditions of DiscoveRx Corporation. A copy of these terms and conditions is available on request. Contact your local DiscoveRx representative for the most current information. This illustration shows an 11-point dose curve with 2 data points each 4 compounds per plate for a total of 8 compounds per eXpress kit. For additional info or further questions regarding the rights conferred with purchase of the Materials, please contact: Licensing Department B. Antibody/Inhibitor Dose Response (96-well plate layout): DiscoveRx Corporation 42501 Albrae Street Fremont, CA 94538 t | 510.979.1415 x 104 e | [email protected] This illustration shows a 10-point dose curve with 2 data points each 4 compounds per plate for a total of 8 compounds per eXpress kit. 14 3 INTENDED USE FREQUENTLY ASKED QUESTIONS PathHunter® eXpress Receptor Tyrosine Kinase (RTK) Functional Assay kits provide Q: I did not see a signal with my control agonist. a robust, highly sensitive and easy-to-use, cell-based functional assay for monitor- A1: There may be differences in agonist purchased from different vendors. ing receptor tyrosine kinase activation, phosphorylation and recruitment of SH2 A2: Confirm that the control agonist used is the same ligand used in the dose containing domain. The eXpress kits contain everything needed for a complete RTK response shown in the provided cell-specific data sheet. experiment including cells, detection reagents, media and plates*. The pre- A3: Please check if appropriate compound diluent was used. validated, frozen eXpress cells have been manufactured for short term use and are provided in a ready-to-assay format that saves time and adds convenience. Assays Q: I did not see a response with my compound. have been designed for 96-well plate analyses. A1: The concentration of DMSO or Ethanol used for dilution is too high. Maintain concentration of the agonist/antagonist diluent at ≤ 1%. *Test compounds are not included and must be provided by the researcher. Please refer to A2: Confirm that the final ligand concentration is correct. Some ligands are DiscoveRx ligands of choice for these assays on Appendix 1. “sticky” and difficult to dissolve. A3: Confirm that the cell line responds to the control agonist. A4: Repeat the experiment using a new lot of control agonist. A5: Please check if appropriate compound diluent was used. TECHNOLOGY PRINCIPLE: PATHHUNTER RECEPTOR TYROSINE KI- NASE ASSAY Q: My cells arrived thawed. Can I use them? A: No. Call technical support for a replacement. Assay-Ready frozen cells are based on DiscoveRx’s PathHunter technology. Path- Hunter cell lines feature novel in vivo applications of Enzyme Fragment Comple- Q: How long is the prepared detection reagent good for? mentation (EFC) technology in which the β-galactosidase (β-gal) enzyme has been A: The working detection reagent solution must be used within 8 hours of mixing. split into two inactive fragments. In the PathHunter assay approach for receptor tyrosine kinases, a small 4 kDa complementing fragment of β-gal (i.e., the ProLink™ Q: What instruments can I use to read the plates? tag) has been fused recombinantly to the intracellular C-terminus of the receptor A: Any bench top luminometer will work with the PathHunter eXpress assays. tyrosine kinase of interest. The larger portion of β-gal, termed EA for ‘enzyme acceptor’, is fused to many different partner proteins containing phospho-tyrosine Q: How long is the signal stable for? binding domains (such as SHC1, SHC2, Grb2, PTPN6, PLCG1 or PLCG2), depending A: The signal is stable for 2-3 hours after addition of detection reagent. on the RTK target. Ligand-induced activation of the receptor causes either homo- or hetero-dimerization of the receptor which results in cross-phosphorylation Q: My cells are floating after the 24 hours incubation. (Figure 1). The SH2-EA fusion protein then specifically binds the phosphorylated A: The cells are not viable, contact technical support for a replacement. receptor resulting in complementation of the two fragments of β-gal and formation of a functional enzyme. β-gal activity is then quantitatively detected using the Q: I ran out the cell plating reagent, can I buy it separately. chemiluminescent substrate in the PathHunter Detection Kit. A: Yes, the reagents are available, please see page 15 for more details. Q: How do I generate a dose curve for my agonist compound? A: Prepare eleven serial 4-fold dilutions of the agonist compound in the desired vehicle of choice. Prepare agonist dilutions such that the concentration is 11X of the final screening concentration (10 µl agonist + 100 µl cells). Pipette all compound dilutions following the plate map on page 14. Q: What Protocol can I use for Antibody studies? A: You can use the inhibitor protocol for a neutralizing antibody. For Activating antibodies, agonist protocol can be used and for Anti-Ligand antibody, a pre- incubation of ligand with the anti-ligand antibody is recommended for 15-30 minutes before the agonist incubation of 3 hours. Figure 1. PathHunter Receptor Tyrosine Kinase Assay Principle. 4 13 QUICK-START PROCEDURE : ANTIBODY/INHIBITOR DOSE RESPONSE STORAGE CONDITIONS PATHHUNTER EXPRESS COMPONENTS REQUIRE MULTIPLE STORAGE TEMPERATURES. OPEN BOXES IMMEDIATELY AND STORE CONTENTS AS INSTRUCTED. Plate 100 µL PathHunter Cells/Well BOX 1: PATHHUNTER EXPRESS CELLS: Store at -80°C* NOTE: PathHunter eXpress cells arrive frozen on dry ice. Cells are delivered in 1 vial containing 1 x 106 Incubate cells in 100 µL of freezing medium. The vial contains sufficient cell numbers to generate (1) 96- 24 or 48 hours well microplate prepared at the seeding density described. @ 37°C *For short term storage (2 weeks or less), store vials at -80°C immediately upon arrival. For storage longer than 2 weeks, place vials in the vapor phase of liquid nitrogen (N2). Do not touch the bottom of the tubes at any time to avoid inadvertent thawing of the cells. If cells are not frozen upon arrival, do not proceed. Contact technical support.