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Updated: A direct comparison of THUNDER™ assay performance with two existing TR-FRET platforms


Bioauxilium is proud to announce the publication of an updated Application Note describing for the first time a comparison among three commercial TR-FRET assay platforms for measuring endogenous phosphoproteins in cellular lysates. The results of this head-to-head study demonstrate superior or comparable performance of THUNDER™ Cell Signaling Assays Kits to that of two existing TR‑FRET platforms while being more cost effective.

  • We compared head-to-head the performance of THUNDER™ with two existing TR-FRET assay technologies.
  • Six endogenous phosphorylated proteins were measured in lysates from cells treated with pathway-specific modulators.
  • THUNDER™ phosphoprotein assays exhibit superior or comparable performance in terms of signal-to-background ratios at a lower cost per well.

The availability of assays capable of measuring target-specific protein phosphorylation in a physiologically relevant cellular context is important for both basic research and drug discovery. Bioauxilium’s THUNDER™ is a new immunoassay platform based on an enhanced TR-FRET technology. The THUNDER™ Cell Signaling Assay Kits are designed to measure endogenous levels of specific intracellular phosphorylated proteins in cell lysates with high sensitivity, specificity, robustness and cost effectiveness (see Figure 1).


Figure 1: THUNDER™ TR-FRET cell signaling assay principle


In this Application Note, we compared head-to-head the performance of THUNDER™ in 384-well plate format with two existing TR‑FRET technologies (Companies A and B) in measuring a panel of six phosphorylated proteins. The three TR‑FRET assay platforms were evaluated for their capacity to measure relative levels of phosphorylated 4EBP1 (T37/T46), AKT pan (S473), ERK1/2 (T202/Y204), p38αβγ (T180/Y182), SLP‑76 (S376), and STAT3 (Y705) in whole-cell lysates from cells treated with pathway-specific modulators. Of note, the THUNDER™ Phospho-SLP-76 and Phospho-STAT3 assay kits have been redeveloped using new antibody pairs to improve their performance.


All assays were conducted according to each manufacturer’s protocols using the standard two-plate transfer protocol for each kit, whereby the cells are seeded, treated and lysed in a 96-well culture plate, and lysates are then transferred to a low-volume 384-well assay plate for protein detection (see Figure 2). Performance metrics were signal-to-background (S/B) ratio, IC50 and EC50, intra-assay variability (%CV), and stability of S/B ratio and IC50/EC50 following overnight incubation.

THUNDER™ assay workflow using the two-plate transfer protocol.
Figure 2: THUNDER™ assay workflow using the two-plate transfer protocol



THUNDER™ assays showed the highest S/B ratios for phospho-AKT pan, phospho-ERK1/2, phospho-p38αβγ and phospho-STAT3. Comparable S/B ratios were obtained for THUNDER™ and
Company A phospho-4EBP1 assay as well as THUNDER™ and Company B phospho-SLP-76 (S376) assay. Company B exhibited the lowest S/B ratios for phospho-AKT pan, phospho-ERK1/2 and
phospho- STAT3. Data obtained for phospho-AKT pan is shown in Figure 3 below.


Figure 3: Head-to-head assessment of the Phospho-AKTpan (S473) assays.


The three TR-FRET technologies exhibited comparable inter-well variability (typical %CV <8%) and sensitivity (IC50 and EC50 values) for all six phosphorylated proteins tested. In addition, all technologies tolerated plate reading after overnight incubation. An assay cost analysis showed that THUNDER™ assay kits are more cost-effective than the other two commercial TR‑FRET technologies.


Collectively, the results of these head-to-head comparisons showed that all THUNDER™ assays tested provide either superior or comparable S/B ratios, and comparable assay pharmacology. In addition, THUNDER™ is more cost effective than the other two TR-FRET assay platforms.

These key advantages, combined with rigorous assay validation using cell lysates from stimulated/inhibited cells, and higher flexibility in terms of kit sizes and formats (detection of either phosphorylated, total or phosphorylated plus total proteins with the same kit), make the THUNDER™ assay platform an alternative of choice for monitoring cellular protein phosphorylation.

See Application Note for more information.