RAS-related protein Rab10 (RAB10) belongs to the RAS superfamily of small GTPases. RAB10 is a key regulator of intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. LRRK2-mediated phosphorylation may cause deficits in endolysosomal trafficking pathways modulated by RAB10, contributing to neurodegenerative disease onset. RAB10 is associated with Alzheimer’s disease and Parkinson’s disease.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-RAB10 (Thr73) assay is a sandwich immunoassay for the quantitative detection of phospho-RAB10 (Thr73) 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
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 phosphorylated 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 phosphoprotein present in the sample.
Phospho-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 Phospho-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.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated 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 RAB10 phosphorylation
RAW 264.7 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 chloroquine for 60 minutes.
After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Thr73) and Total RAB10 levels were evaluated by AlphaLISA SureFire Ultra. 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, chloroquine treatment leads to increased RAB10 phosphorylation with no significant changes in Total RAB10 levels.
A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of chloroquine for 5 hours.
After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Thr73) and Total RAB10 levels were evaluated by AlphaLISA SureFire Ultra. 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, chloroquine treatment leads to increased RAB10 phosphorylation with no change in Total RAB10 levels.
Inhibition of RAB10 phosphorylation
RAW 264.7 cells were seeded in a 96-well plate (80,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of MLi-2 in the presence of 50 ng/mL chloroquine for 3 hours.
After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Thr73) and Total RAB10 levels were evaluated by AlphaLISA SureFire Ultra. 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, MLi-2 treatment resulted in a dose dependent decrease in RAB10 (Thr73) phosphorylation, no decrease in Total RAB10 levels was observed.
RAB10 assay sensitivity - cell lysate dilution
Cell lysate was prepared from A549 cells cultured to confluence in T175 flasks and treated with 250 µg/mL chloroquine for 5 hours. Cells were lysed with 10 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and RAB10 Phospho (Thr73) and Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.
Approximate number of cells/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect RAB10 Phospho (Thr73) in less than 1,000 cells/datapoint.
