| Assay Method Information | |
| | LRRK2 Wild-Type and G2019S Kinase Activity Assay |
| Description: | In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase in kinase reaction buffer was incubated with the test compound (typically at 0 to 30 μM) for 30 minutes before the kinase reaction was initiated by addition of 1.3 mM ATP and 0.4 μM fluorescein-LRRKtide. The reaction mixture (20 μl total volume) was incubated for 3.5 h (for LRRK2 WT) and 3 h (for LRRK2 G2019S) at 30° C., before the reaction was terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 l). The mixture was further incubated for 30 minutes at RT. TR-FRET was measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 s) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 s. The measurement was repeated 30 times for fluorescein and 30 times for terbium emission with a 1000 s time window between repeats. TR-FRET measurements were performed on a Biotek Synergy plate. The TR-FRET signal was calculated as the emission-ratio at 520 nm over 495 nm. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |