Phospho-STAT5 (Tyr694) Cellular Assay Kit

Homogeneous cell-based HTRF® assay kit for measuring endogenous phospho-STAT5 to assess JAK2 inhibitors or as Bcr-abl read out in the JAK/STAT signaling pathway

Cisbio’s homogeneous and robust HTRF® phospho-STAT5 assay kit is designed for the cellular detection and quantification of endogenous STAT5a/b proteins, phosphorylated on Tyr694. Based on TR-FRET technology, the HTRF® phospho-STAT5 assay provides an ideal read out solution, in any cell type, for cytokine mediated JAK/STAT signaling pathway. With an optimized and streamlined mix-and-read, no-wash protocol, the phospho-STAT5 ready-to-use kit contains all reagents you need. By allowing all throughputs, it facilitates drug screening, mechanistic signaling pathway studies, analysis of compound MoA and target research by delivering enhanced convenience and more relevant, reliable results. Let your research flow faster and more efficiently with Cisbio’s smart phospho-protein assays!

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Assay Principle

HTRF® - the homogeneous sandwich immunoassay

Phospho-STAT5 assay is based on a TR-FRET sandwich immunoassay format comprised of two specific anti-STAT5 antibodies, one labeled with Eu3+-cryptate (donor) and the other one labeled with d2 (acceptor). Upon cytokine-depending JAK/STAT pathway activation, STAT5 is phosphorylated on Tyr694. After phosphorylation, the anti-phospho-STAT5 antibody will recognize the phosphorylated residue and the proximity of donor and acceptor will consequently lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for 384-well plate format but can be easily further miniaturized or upscaled. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The phospho-STAT5 assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho-STAT5 can be quantitatively detected using the HTRF® phospho-STAT5 kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection of phosphorylated STAT5, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No washing steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity enabling miniaturization while maintaining a high quality output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

STAT5 cell signaling

The activation of Stat5 proteins, Stat5a and Stat5b, is widely mediated by cytokines, like the IL-2 family, IL-3, IL-4, etc, and other growth factors, allowing rapid delivery of signals from the membrane to the nucleus. Together with other transcription factors and co-factors, the STATs regulate the expression of target genes in a cytokine-specific fashion. In addition to their activation by cytokines, activities of Stat5a and Stat5b, as well as other STAT proteins, are negatively controlled by CIS/SOCS/SSI family proteins. JAK/STAT pathway activation requires ligand binding to the receptor, which in consequence activates the Janus kinases JAKs, a family of intracellular tyrosine kinases. Via phosphorylation of downstream signal transducers and activators of transcription (STAT), STAT5 is forming homo- or heterodimers, becomes thereby active and translocates into the nucleus, where it binds to STAT5 response elements, inducing transcription of specific sets of genes. Upregulation of gene expression by STAT5 dimers has been described for genes involved in cell proliferation, apoptosis, differentiation, inflammation.

The assessment of cellular phospho-STAT5 is an important tool to discover and measure the potency of JAK2 inhibitors, to use a readout of Brc-abl signaling and to generally study the signal transduction in the JAK/STAT pathway.

Product Performance

1. HTRF® assay compared to Western blot

TF1 cells were grown in a T175 flask at 37°C until concentration reached 1.5M/ml of cell culture medium, then stimulated with IL3 for 10min. After removal of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 minutes to lyse the cells. 16µl of serial dilutions (1:2) were analyzed in parallel by HTRF and Western-blot. Note that 128KC corresponds roughly to 32µg of proteins.

By using HTRF® phospho-STAT5 (Tyr694) only 2000 cells are sufficient for minimal signal detection while 8000 cells are needed for a Western blot signal. The HTRF® assay is at least 4-fold more sensitive than the Western blot and shows optimal correlation.

2. HTRF® assay compared to ELISA

100,000 TF1 cells were grown in a T175 flask at 37°C until concentration reached 1.5M/ml of cell culture medium, treated for 30min with 4µl of increasing concentrations of a JAK inhibitor and stimulated with IL3 for 10min. After removal of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 minutes to lyse the cells. Serial dilutions of collected supernatant were analyzed in parallel by HTRF and ELISA. The results displayed in the graph show that for measuring STAT5 phosphorylation HTRF is more sensitive than ELISA, offers a bigger assay window and is more reproducible. Despite a good correlation between ELISA and HTRF, the overall performance of HTRF is more favorable.

3. Pharmacological response on phospho-STAT5 as read out of JAK inhibition by Lestaurtinib in TF1 and HeLa cells

One-plate assay protocol

20,000 TF1cells were plated directly in a 384sv plate suspended in 8µl of cell culture medium treated for 30min with 4µl of increasing concentrations of the JAK inhibitor Lestaurtinib. After removal of cell culture medium, 4µl of 4X supplemented lysis buffer were added and incubated for 30 minutes prior to the addition of the HTRF detection reagents. STAT5 phosphorylation was then measured using the HTRF® phospho-STAT5 (Tyr694) assay.

Two-plate assay protocol

TF1cells were plated in 25µl of cell culture medium treated for 30min with 5µl of increasing concentrations of the JAK inhibitor Lestaurtinib. After removal of cell culture medium, 10µl of 4X supplemented lysis buffer were added and incubated for 30 minutes prior to the addition of the HTRF detection reagents. 16µl of lysates were transferred in a 384sv white plate for analysis of STAT5 phosphorylation. The results clearly demonstrate, that the high sensitivity of the assay allows a 4-fold lower cell quantity to be used without a significant decrease of assay window.

Two-plate assay protocol

100KC HeLa cells were plated in 25µl of cell culture medium treated for 2h with 5µl of increasing concentrations of the JAK inhibitor Lestaurtinib and then stimulated with 20 nM IFN for 10min. After removal of cell culture medium, 10µl of 4X supplemented lysis buffer were added and incubated for 30 minutes prior to the addition of the HTRF detection reagents. 16µl of lysates were transferred in a 384sv white plate for analysis of STAT5 phosphorylation.

According to Diaz et al. PLoS ONE 6(4) (2011) Lestaurtinib Inhibition of the JAK/STAT Signaling Pathway in Hodgkin Lymphoma Inhibits Proliferation and Induces Apoptosis.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
STAT5 phospho-Y694 kit - 500 tests64AT5PEG
STAT5 phospho-Y694 kit - 10,000 tests64AT5PEH
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STAT5 phospho-Y694 kit - 50,000 tests64AT5PEY
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Companion products

DescriptionCat. noProduct insertMSDS
STAT5 phospho-Y694 kit control lysate64AT5TDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF

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