c-kit (CD117) is a receptor tyrosine kinase that binds stem cell factor (SCF) and plays essential roles in hematopoiesis, melanogenesis, and gametogenesis. Upon SCF binding, c-kit undergoes dimerization and autophosphorylation, activating downstream signaling pathways including PI3K/AKT, MAPK, and JAK/STAT to promote cell survival, proliferation, and differentiation. c-kit is critical for the development and maintenance of hematopoietic stem cells, mast cells, and melanocytes. Activating mutations in c-kit are oncogenic drivers in gastrointestinal stromal tumors (GISTs), acute myeloid leukemia, and mastocytosis. Loss-of-function mutations cause piebaldism and certain forms of anemia. Targeted therapies such as imatinib have demonstrated remarkable efficacy in c-kit-driven malignancies, establishing c-kit as a paradigm for precision oncology.
The AlphaLISA SureFire Ultra Human Phospho-c-Kit (Tyr703) is a sandwich immunoassay for the quantitative detection of phospho-c-Kit (Tyr703) 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
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.
SCF induces rapid phosphorylation of c-Kit (Tyr703)
UT-7 and HEL 92.1.7 cells were seeded in a 96-well plate (100K and 400K cells/well respectively) in HBSS + 0.1% BSA. The cells were treated with 100 ng/mL of SCF for the indicated times.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). c-Kit Phospho (Tyr703) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for UT-7 and 16,000 cells for HEL 92.1.7) 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, SCF triggered a rapid phosphorylation of Tyr703 while Total c-Kit levels decreased by 2-fold 30 minutes post treatment.
SCF induces c-Kit phosphorylation in a dose-dependent manner
UT-7 cells were seeded in a 96-well plate (25,000 cells/well) in HBSS + 0.1% BSA. The cells were treated with increasing concentrations of SCF for 5 minutes.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). c-Kit Phospho (Tyr703) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,000 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, SCF triggered a dose-dependent increase in the levels of Phospho c-Kit (Tyr703) with no change to the total levels.
HEL 92.1.7 cells were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and treated with increasing concentrations of SCF for 5 minutes.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). c-Kit Phospho (Tyr703) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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, SCF triggered a dose-dependent increase in the levels of Phospho c-Kit (Tyr703) with no change to total levels.
