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LANCE Classic 3-Component Kinase Assay

Overview


In a LANCE® classic format, a biotinylated peptide or protein substrate is typically used. The phosphorylated site is again recognized by an appropriate Europium-labeled anti-phospho antibody. A SureLight™ streptavidin-APC is added to bind the biotinylated sustrate, to provide an APC acceptor fluorophore. This is a 3-component kinase assay (streptavidin-APC, biotinylated substrate, and Eu-anti-phospho antibody), and will require additional assay development steps. You could also use a tagged substrate (GST, His) with one of our anti-tag antibodies. LANCE® Ultra kinase assays can also be performed as a 4-component assay, if desired.

lance_kinase_classic_main_ASK.jpg
LANCE TR-FRET 3-component kinase assay configuration.

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What do I need to run this assay?


Required reagents available from PerkinElmer:

  • Europium-labeled anti-phospho antibody (in addition to our standard catalog antibodies, you can also Label your own LANCE reagent)
  • SureLight™ streptavidin-APC (or ULight™-streptavidin)
  • 10X LANCE Detection buffer
  • Microplate (we recommend white 384-well OptiPlates™ or ProxiPlates™ )
  • TopSeal™-A adhesive plate seal


Required reagents available from various suppliers:

  • Kinase
  • Biotinylated substrate
  • Kinase buffer (basic kinase buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, and 0.01% Tween 20. Add any essential kinase supplements, e.g., MnCl2, CaCl2, calmodulin, cGMP, lipids etc. as appropriate for your kinase, if needed).
  • EDTA
  • ATP
  • UltraPure water

Instrumentation/equipment:

  • A TRF-capable plate reader

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Products and catalog numbers


View a listing of relevant LANCE reagents with catalog numbers.

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LANCE Classic kinase assay optimizations


Optimization steps

  1. Enzyme titration (control, no enzyme)
  2. Kinetic study (time-course)
  3. Substrate titration (keep ratio of biotin:streptavidin constant)
  4. ATP titration
  5. Biotin:streptavidin ratio titration
  6. Inhibition curve
  7. Z' experiment
  • Enzyme titration/kinetic time-course: different enzyme concentrations are incubated for a short time-period (e.g., 30 min) with a fixed biotin-peptide concentration (e.g., 1 µM) and a non-limiting ATP concentration (no more than 300 µM, as higher ATP concentrations will negatively affect the LANCE signal). Alternatively, one can conduct reaction progression curves (time-course experiments) at each enzyme concentration to select an incubation time that gives both an acceptable S/B ratio and linear kinetics. If one uses 1 µM biotin-peptide (in the 10-µL kinase reaction volume), then the SA-APC concentration should be fixed at 125 nM in the 20 µL total assay volume.
  • Substrate titration: the biotin-peptide is titrated using the optimal enzyme concentration and a non-limiting ATP concentration. Of note, since different biotin-peptide concentrations are used, one must keep the biotin-peptide:SA ratio constant at 4:1. For this, the final SA-APC concentrations are adjusted (e.g., a 500 nM biotin-peptide would require 62.5 nM SA-APC in the 20 µL assay and so on). The biotin-peptide:SA ratio must be kept constant not only to capture all biotin-peptide molecules but also to generate a constant background signal. Keep in mind that the higher the APC concentration, the higher the background signals. Therefore, if one uses a single concentration of SA-APC for all peptide concentrations, the background will be artificially increased for low biotin-peptide concentrations and misleading data will be generated. It is therefore essential to include negative controls (no ATP) for each SA-APC concentration tested in order to correct the signal.
  • ATP titration: the ATP is titrated using the optimal enzyme concentration and the selected biotin-peptide concentration (typically less or equal to its Km value). Since a fixed biotin-peptide concentration is used, a single SA-APC concentration is used (again, keep the 4:1 ratio between biotin-peptide and SA-APC).
  • Biotin:streptavidin titration: different biotin-peptide:SA-APC ratios are tested (e.g., 8:1, 4:1, 2:1) in order to determine the optimal ratio in terms of S/B and cost. Again, include negative controls (no ATP) for each SA-APC concentration tested in order to correct the signal.

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Application notes and other resources for LANCE Classic kinase assays


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