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Homogeneous Kinase Assay Using ATP Lite

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Homogeneous Kinase Assay Using ATP Lite Powered By Docstoc
					 Homogeneous Kinase
  Assay using ATPliteTM

Patricia Kasila and Harry Harney
PerkinElmer Life and Analytical Sciences, Boston MA, USA
    1            Abstract



    ATPlite can be used to measure kinase activity in a homogeneous assay format with-
    out the need for labeled substrates, labeled enzymes or phosphospecific antibodies.
    ATPlite is an Adenosine Triphosphate monitoring system based on firefly (Photinus
    pyralis) luciferase. The assay system is based on the production of light caused by the
    reaction of ATP with added luciferase and D-luciferin. The emitted light is propor-
    tional to the ATP concentration. Kinase enzymes catalyze the transfer of a phosphate
    group from ATP to the substrate. ATPlite is used to measure the depletion of ATP,
    which is directly proportional to the amount of kinase activity. Since protein kinases
    are involved in a wide variety of cellular functions, their role in disease states has
    drawn considerable interest as drug discovery targets. This assay can significantly
    increase throughput and can be easily miniaturized and adapted to HTS robotic
    systems.




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                  2               Introduction



                    Luminescence Assay Systems:
                      • A longer lasting and superior signal for a variety of cell-based and in vitro appli-
                        cations for high throughput and research formats.




                    Features and Benefits:
                      • High sensitivity
                      • Excellent linearity
                      • Simplicity
                      • Can be used for many kinase/substrate combinations
                      • Miniaturizable




                  3               ATPlite Assay System Adapted for
                                  Homogeneous Kinase Assays



                                                                              ATPlite assay system for
                                                                           Homogeneous Kinase assays
                                                                           – Instead of wells containing
                                                                          cells, add substrate and enzyme




                                                                         Luminescence signal for ATPlite
                                                                           can be read on TopCount,
                                                                         MicroBeta, Victor2V, EnVision,
                                                                             ViewLux or any other
                                                                              luminescence reader




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    4            Critical Materials



    ATPlite 300 assay kit (PerkinElmer Catalog # 6016943)
     • 1 X 20 mL of Cell Lysis Solution
     • 1 X 20 mL of Substrate Buffer Solution
     • 3 Vials of Substrate (Luciferase/Luciferin) Solution (Lyophilized)
     • 1 Vial of ATP Standard (Lyophilized)
     • Instruction Booklet
    Substrates
     • PKA Kemptide Substrate – Promega Catalog # V5601
     • Src Substrate Peptide – Upstate Catalog # 12-140
    Enzymes
     • PKA Enzyme – Promega Catalog # V5161
     • Src Enzyme – Upstate Catalog # 14-117
    OptiPlate™- 96, White – PerkinElmer Catalog # 6005290




    5            Methods



    ATPlite Assay Protocol
     • 10 µL Kinase Substrate or Buffer (for negative control)
     • 10 µL ATP
     • 10 µL Enzyme
     • 20 µL Assay Buffer
     • Incubate for 30 min at RT (PKA) or 4 Hours at 30°C (Src)
     • 50 µL ATPlite Substrate solution
     • Dark Adapt for 10 Minutes and Read the Luminescence




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                  6               Results

                                                  ATPlite – PKA Assay




                    The optimal ATP concentration was         The optimal substrate concentration
                    determined by measuring the largest       was determined by measuring the
                    change in luminescence signal in wells    amount of substrate that gives the least
                    with and without substrate.               luminescence signal.




                  7               Results (cont.)

                                                  ATPlite – PKA Assay




                    The optimal kinase concentration was      A dose response curve with staurosporine,
                    determined by measuring the amount of     a kinase Inhibitor was performed with
                    kinase that gave luminescence signal in   the following conditions: 1 µM ATP, 5 µM
                    the linear part of the curve.             substrate, and 0.5 units/well kinase




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    8            Results

                                   ATPlite – Src Assay




    The optimal ATP concentration was          The optimal substrate concentration
    determined by measuring the largest        was determined by measuring the
    change in luminescence signal in wells     amount of substrate that gives the least
    with and without substrate.                luminescence signal.




    9            Results (cont.)

                                   ATPlite – Src Assay




    The optimal kinase concentration was       A dose response curve with stau-
    determined by measuring the amount of      rosporine, a kinase Inhibitor was
    kinase that gave luminescence signal in    performed with the following condi-
    the linear part of the curve.              tions: 0.5 µM ATP, 200 µM substrate,
                                               and 1 unit/well kinase




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               10                 Conclusions



                    • ATPlite can be used for homogeneous kinase assays without the need for substrate
                      or antibody labeling
                    • Phosphorylation by a variety of substrate/kinase combinations can be monitored
                    • Assays can be miniaturized for high throughput screening
                    • Assays can be easily adapted to high throughput screening robotic systems
                    • Due to the simplicity of the ATPlite system, assays can be rapidly developed




                                                  Coming Soon!
                                                  Easylite-Kinase




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