Homogenous time-resolved fluorescence assay (HTRF)

In this format the HTRF assay measures the interaction between GSTSKP2-SKP1 and N-terminal Avi-tagged CKS1. Optimized buffer and detection reagent conditions were 3 nM Tb-anti-GST antibody, 50 nM SA-XL665, in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mg ml⁻¹ BSA, 1% DMSO. Experiments were carried out at GSTSKP2-SKP1 or GSTSKP2_C2-SKP1 concentrations of 1, 5 or 25 nM into which N-AviCKS1 was titrated from 1 to 1000 nM. Reactions were set up in a volume of 20 µl in 384 well plates and incubated for 30 min at 4 °C. Samples were excited using a wavelength of 337 nm and emission spectra measured at 620 nm and 665 nm using a PHERAstar plate reader (BMG Labtech). Binding curves were plotted using GraphPad Prism 6 from which the K_ds were determined.