Description
B-Raf is a serine/threonine-protein kinase and a member of the RAF kinase family, acting downstream of RAS in the MAPK/ERK signaling pathway. It transmits mitogenic signals from the cell membrane to the nucleus, thereby regulating cellular processes such as proliferation, differentiation, and survival. The BRAF V600E mutation results in constitutive kinase activation and is a common driver in melanomas, colorectal cancer, and thyroid carcinoma. Targeted inhibitors against mutant BRAF have demonstrated clinical efficacy, though resistance often develops via MAPK reactivation or bypass mechanisms. Ongoing research explores combination therapies to prolong response and overcome resistance.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-B-Raf (Ser446) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-B-Raf (Ser446) 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.
Inhibition of Phospho B-Raf (Ser446) via PROTAC-mediated degradation
A375 cells were seeded in a 96-well plate (10,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of B-Raf V600E Degrader-1 for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Various targets 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.
B-Raf V600E Degrader-1 specifically degrades B-Raf-V600E in cell lines harboring this mutation, such as A375 cells. As expected, B-Raf V600E Degrader-1 induced a dose-dependent decrease in B-Raf Total levels and a subsequent inhibition of Phospho (Ser446) with no significant changes in Raf-1 Total levels.
Selectivity of Phospho B-Raf assay
Phospho B-Raf (Ser446) levels were assessed in HeLa wild type (WT) and HeLa B-Raf knockout (KO) (Abcam ab265373) cells cultured to confluency in T175 flasks at 37°C, 5% CO2. Each flask was lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were serially diluted in Lysis Buffer, then evaluated for Phospho B-Raf (Ser446) using AlphaLISA SureFire Ultra assay kits.
For the detection step, 10 µL cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was 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.
Phospho B-Raf (Ser446) was only detected in HeLa WT cells, demonstrating assay selectivity.
