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.
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