Leucine-rich repeat kinase 2 (LRRK2, Dardarin, or PARK8) is a large, multifunctional kinase enzyme, classified as a member of the ROCO superfamily. The ROCO family is characterized by the presence of tandem Ras of complex (Roc) G-domain, kinase domains, and carboxy- terminal of Roc (COR) sequence which links them. LRRK2 plays a role in lysosomal function and the innate immunity in the brain (microglia activation) and modulates Tau phosphorylation via GSK-3b. Mutations in LRRK2 are associated with Parkinson’s disease.
The AlphaLISA SureFire Human and Mouse Total LRRK2 Detection Kit is a sandwich immunoassay for the quantitative detection of total LRRK2 in cellular lysates, using Alpha Technology.
Formats
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire kits can be used for
Cellular kinase assays
Receptor activation studies
Screening
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
Activation of Phospho LRRK2 (Ser935) in LPS treated cells
RAW264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 48 hours at 37°C, 5% CO2. The cells were treated with increasing concentrations of LPS for 3 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LRRK22 Phospho (Ser935) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, LPS triggered a dose-dependent increase in the levels of Phospho (Ser935) LRRK2 while Total levels remained unchanged.
Degradation of LRRK2 by PROTAC XL01126
PBMCs were isolated from healthy donors using Ficoll® Plaque Plus and seeded in a 96-well plate (400,000 cells/well) in complete DMEM. Cells were treated with increasing concentrations of XL01126 for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LRRK2 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, XL01126 triggered a dose-dependent decrease in the levels of LRRK2 while ERK levels remained unchanged.
Degradation of LRRK2 by PROTAC XL01126
RAW264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of XL01126 for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LRRK2 and RAB10 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, XL01126 triggered a dose-dependent decrease in the levels of LRRK2 while RAB10 levels remained unchanged.
Versatility of Total LRRK2 assay in various cell lines
Adherent cells were seeded at 50,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 50 µL of Lysis Buffer.
Suspension cells were seeded at 50,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 50 µL of Lysis Buffer. LRRK2 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 10,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
