Validation of THUNDER™ on Molecular Devices SpectraMax® multi-mode readers

Molecular Devices has evaluated the performance of the THUNDER™ Phospho-ERK1/2 (T202/Y204) TR-FRET Cell Signaling Assay Kit on three TR-FRET compatible multi-mode microplate readers: SpectraMax® iD5, SpectraMax® i3x, and SpectraMax® M5e. The results, summarized in an Application Note, showed robust assay performances on all instruments.

Concentration-dependent ERK1/2 (T202/Y204) phosphorylation induced by EGF. Results obtained with SpectraMax iD5 (red), i3x (green), and M5e (blue) readers.

Inhibition of EGF-induced phosphorylation of ERK1/2 (T202/Y204) by Erlotinib. Data obtained with SpectraMax iD5 (red), i3x (green), and M5e (blue) readers.

Highlights

• The THUNDER™ Phospho-ERK1/2 (T202/Y204) Assay Kit was tested in a 96-well format (half-area) using the 2-plate assay protocol.
• Non-starved HEK293 cells seeded at 50,000 cells per well were treated with increasing concentrations of either EGF or erlotinib prior to stimulation with EGF at its EC80 (0.7 nM).
• The plate was then read on the SpectraMax iD5, SpectraMax i3x, and SpectraMax M5e readers using optimized settings.
• Concentration-responses curves generated EC50 and IC50 values ranging between 0.28 and 0.41 nM for EGF, and between 42 and 63 nM for erlotinib. These values are consistent with published data.
• Overall, the THUNDER Phospho-ERK1/2 (T202/Y204) assay showed robust TR-FRET signals, wide dynamic ranges, high S/B ratios, and the expected pharmacology on SpectraMax iD5, i3x, and M5e readers.
• Of note, comparable data were obtained using optimized HTRF® settings, demonstrating the robustness of the THUNDER™ cellular assay.
• Molecular Devices SpectraMax® readers are therefore certified for use with THUNDER™.