This HTRF kit enables both SAPK/JNK and p38 MAPK pathway readout, measuring ATF2 modulation phosphorylated at Threonine-71.

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  • Ease-of-use
  • Highly specific
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This HTRF kit enables both SAPK/JNK and p38 MAPK pathway readout, measuring ATF2 modulation phosphorylated at Threonine-71.

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Overview

ATF2 (Activating transcription factor 2) is a transcription factor activated by phosphorylation on threonin residues by JNK/SAPK. The phospho-ATF2 (Thr71) kit enables the quantitative detection of transcription factor ATF2 modulation phosphorylated at Threonine-71. ATF2 is also involved in PI3K/AKT, TNF, and estrogen signaling along with insulin secretion, thyroid hormone release, substance dependence, viral carcinogenesis and infectious diseases. The simple add-and-read protocol features no wash steps for a faster analysis in biological applications like oncology, diabetes or inflammation.

Phospho-ATF2 (Thr71) kit assay principle

The Phospho-ATF2 (Thr71) assay measures ATF2 when phosphorylated at Thr71. Contrary to 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, the other one 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 independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and 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 kit 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 adding Phospho-ATF2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-ATF2 kit 1-plate assay protocol

Detection of Phosphorylated ATF2 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.

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.

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 & 60 minutes. After lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-ATF2 (Thr71) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

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 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL 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.

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.

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert ATF2 P-T71 Kit / 63ADK015PEG-63ADK015PEH

63ADK015PEG-63ADK015PEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Safety Data Sheet ATF2 P-T71 Kit / 63ADK015PEG

63ADK015PEG - Safety Data Sheet

Safety Data Sheet ATF2 P-T71 Kit / 63ADK015PEH

63ADK015PEH - Safety Data Sheet

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