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Assay Method Information |
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| | Homogeneous Time-Resolved Fluorescence (HTRF) Enzyme Assay |
| Description: | HTRF relies on fluorescence resonance energy transfer (FRET) between the donor, a europium cryptate (EuK), and the acceptor, the light harvesting protein allophycocyanin (XL-665). The donor, EuK (Lance), was coupled to an anti-phospho-ATF2 antibody that specifically binds Thr71-phosphorylated ATF2 and the XL-665 acceptor was supplied coupled to streptavidin to bind the biotinylated substrate GST-ATF2. Thus, when the biotinylated ATF2 is phosphorylated by JNK3 or p38, EuK and XL665 are brought into close proximity allowing the FRET to take place following laser excitation of EuK at 337 nm. The HTRF Discovery reader (Packard) detects emissions at 620 nm (non-specific B-counts) and at 665 nm (specific A-counts). The HTRF signal is calculated as a ratio of the A-counts to B-counts. The signal produced is proportional to the amount of JNK3 or p38 kinase activity present during the kinase reaction phase. |
| Affinity data for this assay | |
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