Besifloxacin Hydrochloride (BOL-303224-A) is a fourth-generation fluoroquinolone antibiotic.

20 ng of purified human DDK, together with increasing concentrations of each DDK inhibitor is pre-incubated for 5 min. Then 10 μCi (γ)-32P ATP and 1.5 μM cold ATP are added in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT and incubated for 30 min at 30°C. The proteins are denatured in 1X Laemmli buffer at 100°C followed by SDS-PAGE and autoradiography on HyBlot CL film. DDK kinase activity is indicated by Auto-phosphorylation of DDK. 32P-labeled bands are quantified using ImageJ and the IC50 values are calculated using GraphPad.