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TKXtra DataFile: Homogenous tyrosine kinase detection using High Efficiency Fluorescence Polarization

mperature 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 conce
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