In JoVE Journal: Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells using THUNDER™ TR-FRET

BioAuxilium has published an article in the J Vis Exp (JoVE) Journal (Biochemistry). In this paper, we describe in detail protocols for the simple, sensitive, robust, and cost-effective measurement in a 384-well format of endogenous phosphorylated STAT1/3/4/5/6 proteins, together with total STAT1/3/5/6, in cell lysates using the THUNDER™ TR-FRET platform.

THUNDER™ TR-FRET assay workflow.

Detection of phospho-STAT5 (Y694/Y699) and total STAT5 modulation in A431 cells.

In brief:

• We measured the intracellular levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641), together with total STAT1, STAT3, STAT5, and STAT6, in cell lysates from adherent or suspension cells.
• The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). It consists of 3 steps: cell treatment, cell lysis, and protein detection using TR-FRET.
• The no-wash assay protocols are flexible, use a low-volume sample (15 µL), require only one reagent addition step, and can be adapted to low-throughput and high-throughput applications.
• In the two-plate assay protocol, lysates are transferred to a white 384-well detection plate, whereas in the one-plate protocol, all steps are conducted in the same white 384-well detection plate (all-in-one-well protocol).
• Each phospho-STAT sandwich immunoassay was validated under optimized conditions with known agonists and inhibitors and generated the expected pharmacology and Z’-factor values.
• As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches like ELISA.
• This suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.
• Given its simplicity, specificity, sensitivity, reproducibility, and cost-effectiveness, the THUNDER™ TR-FRET platform represents an attractive alternative to traditional immunoassays, both in an academic setting and in industrial laboratories for HTS applications.

For more information, see the full article here: doi: 10.3791/62915