| Solution Information | help | |
| Enzyme: | Mitogen-activated protein kinase 10 | |
| inhibitor: | BDBM42816 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary biochemical high-throughput screening assay for inhibitors of the c-Jun N-Terminal Kinase 3 (JNK3)," (PubChem AID 746). In this assay phosphorylation of the JNK3 substrate Activating Transcription Factor 2 (ATF2) is measured using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). The assay uses biotinylated ATF2 (b-ATF2), which binds both XL-665-labeled streptavidin (SA-XL-665) and a europium-labeled antibody (Eu-Ab) that specifically recognizes the phosphorylated form of b-ATF2. Upon b-ATF2 phosphorylation by JNK3, Eu-Ab binds the phosphorylated b-ATF2. The addition of SA-XL-665 brings the FRET donor (Eu-Ab) and acceptor (SA XL-665) pair into close proximity, allowing FRET to occur. As designed, a compound that inhibits JNK3 activity will prevent JNK3-mediated phosphorylation of b-ATF, Eu-Ab binding t | |
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