Validation of THUNDER™ on the HIDEX Sense multimode reader

HIDEX has assessed the performance of THUNDER™ Phospho-ERK1/2 (T202/Y204) TR-FRET Cell Signaling Assay Kit and THUNDER™ Phospho-STAT4 (Y693) TR-FRET Cell Signaling Assay Kit on the HIDEX Sense multi-mode microplate reader. The results showed robust assay performances with a variety of instrument settings.

Titration of the Phospho-ERK1/2 Control Lysate. Donor data (Europium chelate) were obtained with an emission filter at either 616 or 620 nm.

Titration of the Phospho-STAT4 Control Lysate. Donor data (Europium chelate) were obtained with an emission filter at either 616 or 620 nm.

Highlights

• The THUNDER™ Phospho-ERK1/2 (T202/Y204) and Phospho-STAT4 (Y693) Assay Kits were tested by conducting a titration of the control lysate provided with the kits (QC test described in the TDS). Samples were tested in a low-volume 384-well plate.
• The plate was then read on the HIDEX Sense using a range of settings.
• Overall, the QC tests showed the expected and robust TR-FRET signals, dynamic ranges, and S/B ratios.
• Of note, comparable data were obtained using optimized HTRF® settings, demonstrating the robustness of the THUNDER™ cellular assays.
• Based on these results, the recommended optimal settings for THUNDER™ are the following:
  – Excitation filter: 330 (80) nm; Emission donor: 620 (10) nm; Emission acceptor: 665 (7.5) nm
  – Number of flashes: 200
  – Delay: 100 µsec
  – Reading time: 400 µsec
  – Lamp power: Medium

  – Focus: default

• The HIDEX Sense multimode reader is therefore certified for use with THUNDER™.