Materials & Methods
Reagents: SNAP-Lumi4-Tb, anti-mouse Fc-d2, anti-human Fc-d2, anti-cMyc-d2, anti-His-d2, SNAP-RTK plasmids, and the labeling medium were from Cisbio Bioassays. Lipofectamin 2000 was purchased from Invitrogen, and the cell-dissociation buffer was from Sigma-Aldrich. Anti-RTK antibodies were obtained from different sources.
Covalent labeling of SNAP-RTK cells: A solution of Tag-lite SNAP-Lumi4-Tb substrate at 100 nM was prepared in Tag-lite labeling medium. After aspiration of the cell culture medium, the SNAP-Lumi4-Tb solution was added to the cells expressing the SNAP-RTK in the T175 flask, followed by one hour incubation at 37ºC. The cells were then washed four times to remove the excess of SNAP-Lumi4-Tb and detached using the cell-dissociation buffer. The cells were frozen under 1 million cells/vial in culture medium and 10% DMSO.
Antibody binding assay: The SNAP-Tb labeled cells were thawed and washed with PBS, then re-suspended in labeling buffer. Binding assays were performed in a total volume of 20 μL in 384 small volume white plates. 10,000 cells/well were incubated for two hours at room temperature with two different concentrations of primary antibodies (10 and 40 nM). Red labeled antispecies antibodies or Red-labeled antitag antibodies were added at a fixed and saturating concentration (100 nM) for the detection.
Erk phosphorylation assay: SNAP-EGFR1 expressing cells were dispensed in 384 sv plates and pretreated or not with Cetuximab or erlotinib (tyrosine kinase inhibitor) for two hours, at 37°C, then stimulated with EGF for 10 minutes. After cell lysis, phosphorylated ERK was quantified by HTRF using the Cellul’erk kit.