HTRF Human Phospho-LRRK2 Ser1292 Detection Kit, 500 assay points

The Phospho-LRRK2 (Ser1292) assay measures LRRK2 when phosphorylated at Ser1292. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation leads to 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.

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Activation buffer followed by Phospho-LRRK2 (Ser1292) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

HEK293 cells were transfected in a T175 flask with a plasmid encoding the human mutant LRRK2 G2019S protein for 24 hours and then treated with 100 nM Calyculin-A for 30 minutes. The cells were lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at room temperature under gentle shaking.

The competition assay was carried out in a 384-well low volume white microplate by dispensing 12 µL of cell lysate (previously diluted 20-fold in supplemented lysis buffer), 2 µL of each peptide (serially diluted in supplemented lysis buffer), 2 µL of Activation buffer, and 4 µL of the HTRF Phospho-LRRK2 (Ser1292) detection reagents. The HTRF signal was recorded on an EnVision Nexus reader after an overnight incubation at room temperature.

A dose-dependent inhibition of the HTRF signal was obtained with the LRRK2 phospho-Ser1292 peptide, while the signal remained unchanged in the presence of the other peptides. These results demonstrate that the HTRF assay specifically detects the phosphorylated Ser1292 residue on LRRK2.

HEK293 cells were transfected in a T175 flask with a plasmid encoding the human mutant LRRK2 G2019S protein for 24 hours, and then treated with 100 nM Calyculin-A for 30 minutes. The cells were lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 14 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 2 µL of Activation buffer and 4 µL of HTRF Phospho-LRRK2 (Ser1292) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Phospho-LRRK2 (Ser1292) assay, 1560 cells/well were enough to detect a significant signal, while 6250 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Phospho-LRRK2 (Ser1292) assay was 4 times more sensitive than the Western Blot technique.

Under physiological conditions, LRRK2 activation and localization are regulated by the Rab GTPase Rab29. Rab29 recruits LRRK2 to the membranes of different organelles (trans-Golgi network, mitochondria, phagosomes, and damaged lysosomes) under specific stimuli. This recruitment leads to the stimulation of LRRK2 kinase activity and a subsequent increase in LRRK2 autophosphorylation at Ser1292. Activated LRRK2 phosphorylates a subset of membrane-associated Rab GTPases (Rab3, Rab8, Rab10, Rab12, Rab35, Rab43) and interacts with other downstream proteins, which together regulate a variety of cellular processes such as endocytosis and vesicular trafficking, microtubule dynamics, the autophagy-lysosomal pathway (ALP), Golgi and mitochondrial function, as well as α-synuclein homeostasis.

Pathogenic LRRK2 mutations, such as the common G2019S mutation located in the kinase domain, increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that contributes to the Parkinson’s Disease (PD) phenotype.