( A Sensitive Robust Homogenousa...)TKXtra DataFile: Homogenous tyrosine kinase detection using High Efficiency Fluorescence Polarization

A Sensitive, Robust, Homogenous,and Rapid Assay
Uses High Efficiency Fluorescence Polarization (HEFP) detection method
Assays a broad range of tyrosine kinases
Requires no separation or wash steps
Available in a kit and in bulk packaging

Introduction
Screening for tyrosine kinase activity can be accelerated using High Efficiency Fluorescence Polarization (HEFP) assays. Molecular Devices TKXtra HEFP Assay Reagents provides a sensitive and rapid assay that is applicable for detecting the activity of numerous tyrosine kinases. For convenience and economy, the reagents can be purchased as a kit for assay evaluation and in bulk for high-throughput screening.

Intended Use
This product is designed to determine an increase or decrease in activity of tyrosine kinase in solution.

Principle of the Assay
The TKXtra system measures the activity of tyrosine kinases via a competitive immunoassay for the product of enzyme activityphosphotyrosine (pY)-containing peptides. The TKXtra procedure is a two-step process: first a kinase reaction is run, then a HEFP immunoassay is performed on the reaction products to quantify the amount of phosphopeptide formed.¹

Kit Contents
The kit contains anti-phosphotyrosine antibody, fluorescein-labeled tracer, phosphotyrosine calibrator, and assay buffer. When used as suggested, each kit provides sufficient reagents for 1600 40-L assays.

For assay volumes between 440 L, HE microplates are recommended for optimal results.

Assay Conditions
Samples containing phosphopeptides produced by the tyrosine kinase assay are mixed with fluorescein-labeled tracer and anti-phosphotyrosine antibody. The samples are incubated at ambient temperature and analyzed after 30 minutes with the same performance, using any instrument in the Criterion family.

Applications
Monitoring the activity of receptor and non-receptor tyrosine kinases

Screening of agonists or antagonists of tyrosine kinases

The TKXtra assay is capable of detecting a number of kinases. For example v-abl, EGF receptor, insulin receptor, and c-src have all been detected with good sensitivity.

Assay Performance
TKXtra calibration curve: A calibration curve was performed in a 384-well microplate. The calibrator is a phosphotyrosine peptide that closely mimics the assay response to tyrosine kinase products. This curve illustrates the nanomolar sensitivity of the TKXtra kit.

Optimization of β−Insulin receptor kinase (β-IRk) tyrosine kinase: Titration curves were performed in a 384-well microplate using varying amounts of enzyme and three concentrations of ATP. The reaction mixture contained 5 L each of PGT (2 M final concentration), ATP and 8 L of β-IRk enzyme. After 2 hours of incubation at ambient temperature, the reaction was stopped by adding 2 L of 200 mM EDTA solution prior to running the HEFP portion of the TKXtra assay.

The data show that under the defined conditions, an assay for the β-IRk could be optimized using 0.031 nM β-IRk and 10250 M ATP.

Inhibition of β-IRk activity by staurosporine: The inhibition of the insulin receptor tyrosine kinase by staurosporine was performed in a 384-well microplate with a 20-L sample volume. The reaction mixture contained final concentrations of the following components: 2 M PGT, 25 M ATP, 0.1 nM β-IRk, and various concentrations of staurosporine. In the presence of 0.1 nM β-IRk and 25 M ATP, the IC50 value is 90 nM with the TKXtra kit, a figure that agrees with literature values.²

¹ Seethala, R. and Menzal, R. Anal. Biochem. 255:257262 1998

² Fujita-Yamaguchi, Y. and Kathuria, S. Biochem. Biophys. Res. Commun. 157:955962 1988

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