WH-4-023

WH-4-023 shows little activity on p38 and KDR. ^[1]

• Lck Kinase Assay

  The Lck HTRF kinase assay involves ATP-dependent phosphorylation of a biotinylated substrate peptide of gastrin in the presence or absence of inhibitor compound. The final concentration of gastrin is 1.2 μM. The final concentration of ATP is 0.5 μM (Km app = 0.6 ± 0.1 μM), and the final concentration of Lck (a GST-kinase domain fusion (AA 225−509)) is 250 pM. Buffer conditions are as follows:  50 mM HEPES pH = 7.5, 50 mM NaCl, 20 mM MgCl₂, 5 mM MnCl, 2 mM DTT, 0.05% BSA. The assay is quenched and stopped with 160 μL of detection reagent. Detection reagents are as follows:  Buffer made of 50 mM Tris, pH = 7.5, 100 mM NaCl, 3 mM EDTA, 0.05% BSA, 0.1% Tween20. Prior to reading, Streptavidin allophycocyanin (SA-APC) is added at a final concentration in the assay of 0.0004 mg/mL, along with europilated anti-phosphotyrosine Ab (Eu-anti-PY) at a final conc of 0.025 nM. The assay plate is read in a Discovery fluorescence plate reader with excitation at 320 nm and emission at 615 and 655 nm. Assays for other kinases are done in a similar way as described above, varying the concentrations of enzyme, peptide substrate, and ATP added to the reaction, depending on the specific activity of the kinase and measured Kms for the substrates.

• Animal Models

  C57BL/6 mice

• Dosages

  100 mg/kg

• Administration

  i.v.