Fusion α plate reader

The binding affinity of biotinylated GST-Ei24N, GST-CanRch1 or GST alone to His₆-tagged IMPs was determined using the bead-based AlphaScreen assay (PerkinElmer), as described previously (Wagstaff and Jans, 2006 (link)). In brief, 30 nM of biotinylated GST-Ei24N, GST-CanRch1, or GST alone was incubated with increasing concentrations of His₆-tagged IMPs. After incubation with nickel-chelating acceptor and streptavidin-coated donor beads, results were read on a Fusionα plate reader (PerkinElmer). Triplicate values were averaged and sigmoidal titration curves (three-parameter sigmoidal fit) were plotted using the SigmaPlot graphing program (Systat Software Inc.) to determine the dissociation constant (K_d) and maximal binding (B_max) value. It should be noted that the sensitivity of the AlphaScreen binding assay results in lower estimated K_d values than various other assays (e.g., Catimel et al., 2001 (link); Forwood and Jans, 2002 (link); Harreman et al., 2003a (link),b (link); Fulcher et al., 2010 (link)), but relative/comparative values are comparable (e.g., see the legend for Table 1).

Initial ERK1/2 phosphorylation time course experiments were performed to determine the time at which ERK1/2 phosphorylation was maximal after stimulation by each ligand. Cells were seeded into transparent 96-well plates at 20,000 cells per well and grown for over 8 h. Cells were then washed once with PBS and incubated in serum-free DMEM at 37 °C overnight to allow FBS-stimulated phosphorylated ERK1/2 levels to subside. Cells were then stimulated for 25 min without or with antagonist, followed by a 5-min agonist incubation at 37 °C in 5% CO₂. For all experiments, 10% (vol/vol) FBS was used as a positive control, and vehicle controls were also performed. The reaction was terminated by removal of drugs and lysis of cells with 100 μL of SureFire lysis buffer (TGR Biosciences), and 5 μL of this lysate was added in a 384-well white ProxiPlate (PerkinElmer). A mixture of SureFire activation buffer, SureFire reaction buffer, and AlphaScreen beads was prepared in a ratio of 100:600:3 (vol/vol/vol) and added to the lysate for a lysate/mixture ratio of 5:8 (vol/vol). Plates were incubated for 1–1.5 h at 37 °C before the fluorescence signal was measured on a Fusion-α plate reader (PerkinElmer) using standard AlphaScreen settings.