Phospho-ATF2 (Thr71) Cellular Assay Kit

Homogeneous cell-based assay for phospho-ATF2 as a readout of JNK1 or p38MAPK

Cisbio's cell-based homogeneous HTRF® phospho-ATF2 assay (Thr71) enables both SAPK/JNK and p38 MAPK pathway readout, giving quantitative detection of the modulation of the transcription factor ATF2, phosphorylated on Threonine 71. ATF2 is also involved in PI3K/AKT, TNF and estrogen signaling, and amongst others ATF2 plays a role in insulin secretion, thyroid hormone release, substance dependence, viral carcinogenesis and infectious diseases. The kit is suitable for analysing pharmacological questions in cellular models. The simple add-and-read protocol, with no wash steps for a faster analysis, facilitates a wide variety of biological applications from basic reasearch to high throughput screening in oncology, diabetes, inflammation, cardiovascular, virology and CNS areas.

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How it works

Phospho-ATF2 (Thr71) assay principle

The Phospho-ATF2 (Thr71) assay measures ATF2 when phosphorylated at Thr71. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-ATF2 (Thr71) assay uses 2 labeled antibodies, one with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-ATF2 (Thr71) assay principle

Phospho-ATF2 (Thr71) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-ATF2 (Thr71) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored

Phospho-ATF2 (Thr71) 1-plate assay protocol

Phospho-ATF2 (Thr71) 1-plate assay protocol

Detection of Phosphorylated ATF2 (Thr71) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Phospho-ATF2 (Thr71) 1-plate assay protocol

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.

Assay validation

1. Pharmacological validation of phospho-ATF2 (Thr71) in NIH3T3 cells

Mouse NIH3T3 cells were seeded at different cell densities in 96-well microplates, then stimulated with increasing concentrations of anisomycin for 30 minutes. Following the 2-plate assay protocol, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-ATF2 (Thr71) detection reagents. The HTRF signal was recorded after an overnight incubation.

Image showing the importance of Pervanadate to detect Phospho-ATF2 Thr71

Image of the validation of the Cisbio HTRF phospho and total ATF2 on human HEK293A cells treated with anisomycin

2. Anisomycin dose-response and time-course in HEK293A cells

Human HEK293A suspension cells were seeded at 100,000 cells/well in a 96 well half area plate, and incubated for 24h at 37°C, 5% CO2. Then cells were stimulated with different concentrations of anisomycin for 15, 30 or 60 minutes. After lysis, 16µL of lysate were transferred into a 384-well low volume white microplate and 4µL of the HTRF phospho-ATF2 (Thr71) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

3. HTRF phospho-ATF2 cellular assays compared to Western Blot

The human NIH3T3 cell line was seeded in a T175 flask and incubated a 37°C, 5% CO2 until confluency.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16µL of each dilution were transferred into a low volume white microplate before the addition of 4µL of HTRF phospho-ATF2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF Phospho-ATF2 T71 assay, 3,000 cells/well were sufficient to detect a signal while 6,125 cells were needed using Western Blot with an ECL detection. These results demonstrate that the HTRF phospho-ATF2 assay is 2 times more sensitive than the Western Blot.

Image of the comparison between Western blot and HTRF using phospho-ATF2 on NIH3T3-cells

Simplified pathway

Function and regulation of ATF2

In response to cellular stress, such as exposure to genotoxic agents, cytokines, or UV irradiation, SAPK/JNK and p38 MAP kinases are activated and phosphorylate the transcription factor ATF-2 on residues Thr69 and Thr71. Once in the nucleus, phospho-ATF2 binds to cAMP response element (CRE) or to Activator Protein-1 (AP-1) response elements, and regulates the transcription of genes involved in cell growth, survival, and DNA damage response.

ATF2 signaling pathway

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
ATF2 phospho-T71 kit - 500 tests63ADK015PEG
ATF2 phospho-T71 kit - 10,000 tests63ADK015PEH
ATF2 phospho-T71 kit - 50,000 tests63ADK015PEY
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Companion products

DescriptionCat. noProduct insertMSDS
ATF2 phospho-T71 kit - control lysate63ADK015TDA
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