| Assay Method Information | |
| | LRRK2 Wild-Type and G2019S Kinase Activity Assay |
| Description: | The LRRK2 kinase was obtained from Invitrogen (Life Technologies Corporation) and comprises residue 970 to 2527 of the full length human wild-type LRRK2 kinase, or a similar sequence with the G2019S mutation. As discussed above, this mutation increases the kinase activity relative to the wild type. The kinase reactions were performed in a 20 uL volume in 384-well plates. The kinase reaction buffer consisted of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT.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 uM) for 30 minutes before the kinase reaction was initiated by addition of 1.3 mM ATP and 0.4 uM fluorescein-LRRKtide. The reaction mixture (20 ul 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 ul). 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 us) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 us. The measurement was repeated 30 times for fluorescein and 30 times for terbium emission with a 1000 us 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 | |
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