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HTRF Human & Mouse Phospho-ATF2 (Thr71) Detection Kit, 500 Assay Points

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

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Feature Specification
Application Cell Signaling
Sample Volume 16 µL

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

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 500 Assay Points
Part #:
63ADK015PEG
List Price
USD 2,147.00
Unit Size: 10,000 Assay Points
Part #:
63ADK015PEH
List Price
USD 12,490.00

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.

Specifications

Application
Cell Signaling
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 1
Lysis Buffer 2
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
TR-FRET
Therapeutic Area
Infectious Diseases
Metabolism/Diabetes
Unit Size
500 Assay Points

Video gallery

How it works

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-how-it-works-phospho-atf2-kit-assay-principle

 

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-how-it-works-phospho-atf2-kit-2-plate-assay-protocol

 

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.

phospho-atf2-kit-1-plate-assay-protocol

 

Assay validation

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.

assay-validation-atf2-phospho-y542-1

 

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.

assay-validation-atf2-phospho-y542-2

 

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.

assay-validation-atf2-phospho-y542-3

 

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.

phospho-pathway-atf2-phospho-y542-kit

 

Resources

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Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

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