RAB12 is a member of the RAB family of small GTPases that regulate intracellular vesicle trafficking and autophagy. RAB12 localizes to recycling endosomes and lysosomes, modulating the trafficking and degradation of membrane proteins, including the transferrin receptor and amino acid transporters. It plays a role in nutrient sensing, mTORC1 regulation, and stress-induced autophagy. In cancer, altered RAB12 expression is associated with dysregulated autophagic flux and resistance to chemotherapy. It also contributes to glioma cell invasion and migration. As a regulator of vesicle transport and cellular metabolism, RAB12 is emerging as a context-dependent modulator in cancer and metabolic disease.
The AlphaLISA SureFire Ultra Human and Mouse Total RAB12 Detection Kit is a sandwich immunoassay for the quantitative detection of total RAB12 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
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
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 RAB12 phosphorylation in endogenous cell systems
RAW 264.7 cells and HepG2 cells were seeded in a 96-well plate (80,000 cells/well and 50,000 cells/well, respectively) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Chloroquine for 4 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). RAB12 Phospho (Ser106) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 (RAW 264.7) or 5,000 (HepG2) 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 triggered a dose-dependent increase in the levels of Phospho RAB12 (Ser106) while Total RAB12 levels remained unchanged.
Inhibition of RAB12 phosphorylation in MLi2 treated cells
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 MLi2 with 100 µg/mL Chloroquine for 4 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). RAB12 Phospho (Ser106) 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, MLi2 triggered a dose-dependent decrease in the levels of Phospho RAB12 (Ser106) without any change to Total RAB12 levels.
Versatility of Total RAB12 assay in various cell lines
Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer.
RAB12 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.
RAB12 is ubiquitously expressed in a range of common cell lines.
