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LANCE® Ultra assays are based on time-resolved fluorescence resonance energy transfer (TR-FRET). They use a proprietary europium chelate donor dye, W-1024 (Eu), with ULight®, an innovative small molecular weight acceptor dye with a red-shifted fluorescent emission. In kinase assays, the binding of a Eu-labeled anti-phospho-substrate antibody to the phosphorylated ULight-labeled substrate brings donor and acceptor molecules into proximity. After irradiation of the kinase reaction at 320 or 340 nm, the energy from the Eu donor is transferred to the ULight acceptor dye which, in turn, generates light at 665 nm. The intensity of the light emission is proportional to the level of ULight-substrate phosphorylation.
Focusing on TK EphA4
Tyrosine Kinase (TK) EphA4 is part of a 14-member family of receptor tyrosine kinases with functions in intercellular communication, migration, patterning and angiogenesis. Eph's are implicated in development of tumor vasculature and intercellular contacts required for metastasis. Several members are overexpressed in cancers. Soluble forms (competitive receptors) have shown some anti-tumor activity, and extracellular domains have been used as tumor-specific antigens.
EphA4 has been cropping up in the literature with EphA4 activity linked to numerous disorders in oncology, neurology, even orthopedics and beyond (See references below). As EphA4 is increasingly discovered to be a significant tyrosine kinase beyond its usual association in neural pathways, uncovering new methods and reagents for measuring its activity becomes a paramount need.
New LANCE Ultra ULight-TK Substrate
Announcing the new ULight-TK peptide for the LANCE Ultra TR-FRET assay platform. This new substrate is ideal for the vast majority of receptor and cytoplasmic tyrosine kinases as demonstrated by its use in the EphA4 assay described here. The Eu-anti-phosphotyrosine (PT66) was used to capture the peptide’s phosphorylated tyrosine residues. Optimization of the assay included kinase and ATP titration and time-course. We also determined the IC50 value of EphA4 for staurosporine and assessed assay robustness by Z’-factor determination. The optimized EphA4 reaction includes 120 pM EpHA4, 35 µM ATP and 50 nM ULight-TK peptide in a 10 µL assay reaction including 50 mM HEPES pH 7.5, 10 MgCl2, 1 mM 1 mM EGTA, 2 mM DTT and 0.1% Tween-20. After completion of the kinase reaction, EDTA and Eu-anti-phosphotyrosine (PT66) were added to final concentrations of 10 mM and 2 nM, respectively, in LANCE Detection buffer in a final volume of 20 µL.
Our data indicate a Km app value for ATP of 34 µM and IC50 value for staurosporine of 117 µM. A high Z’-factor of 0.79 demonstrates the LANCE Ultra EphA4 assay robustness.
Enzyme Titration and Time Course
The EphA4 enzyme was incubated at concentrations ranging from 120 to 960 pM with 50 nM ULight-TK peptide and 200 mM ATP. Kinase reactions were terminated by the addition of EDTA after 0 to 120 min at 23°C. Eu-anti-phosphotyrosine (PT66) was added to a concentration of 2 nM and signal was read after 1 h with the EnVision® Multilabel Plate Reader.
Experiment 2: ATP Titration
Serial dilutions of ATP ranging from 10 nM to 1 mM were added to 120 pM EphA4 enzyme and 50 nM of ULight-TK Peptide. Kinase reactions were terminated after 60 min at 23°C by the addition of EDTA. Eu-anti-phosphotyrosine (PT66) was added to a concentration of 2 nM and signal was read after 1 h with the EnVision Multilabel Plate Reader.
Experiment 3: Enzyme Inhibition Curve
Serial dilutions of Staurosporine ranging from 30 pM to 10 µM (final concentrations in 2% DMSO) were incubated with 120 pM EphA4 enzyme, 50 nM ULight-TK peptide and 35 µM ATP. Kinase reactions were terminated after 60 min at 23°C by the addition of EDTA. Eu-anti-phosphotyrosine (PT66) was added to a concentration of 2 nM and signal was read after 1 h with the EnVision Multilabel Plate Reader.
Experiment 4: Z’-factor Determination
EphA4 enzyme at 120 pM was incubated with 50 nM ULight-TK Peptide, 35 µM ATP and 10 µM staurosporine (final concentrations in 2% DMSO). Kinase reactions were terminated after 60 min at 23°C by the addition of EDTA. Eu-anti-phosphotyrosine (PT66) was added to a final concentration of 2 nM and signal was read after 1 h with the EnVision Multilabel Plate Reader.
Homogenous and stable peptides for better detection
The ULight-TK peptide was developed to provide the researcher with a universal peptide substrate for both Receptor and Cytoplasmic Tyrosine Kinases. The TK peptide has been designed to be homogeneous in size and more stable in the assay than the generic substrates, ULight- poly GT and polyGAT, that is available today. We have validated that this TK peptide serves as an effective peptide substrate for 49 receptor- and 34 cytoplasmic-tyrosine kinases using the LANCE Ultra assay platform. Based on the measured signal/background ratio (determined from signal at 665 nm, +/- ATP) the new ULight-TK peptide (Cat. No. TRF0127) out performed the generic peptide substrates 66% of the time. Please consult the LANCE Ultra Selection Guide for Tyrosine kinases to identify the best substrate for your enzyme at:
Contact your local PerkinElmer account manager to learn more and how to incorporate this new reagent in your kinase screening efforts.
Validated on EnVision
These PerkinElmer assays were run
on the EnVision® Multilabel Plate Reader with TRF reader. EnVision Multilabel Plate Readers are fast, sensitive and versatile benchtop readers that deliver optimized performance in every application and for every label.
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