B-Raf is a serine/threonine protein kinase and member of the RAF family that functions as a critical component of the RAS-RAF-MEK-ERK signaling cascade. B-Raf is activated by RAS-GTP binding, promoting dimerization and kinase activation, leading to phosphorylation of MEK1/2. The V600E mutation in B-Raf, found in approximately 50% of melanomas, results in constitutive kinase activity and oncogenic transformation. Other B-Raf mutations can paradoxically activate wild-type RAF through enhanced dimerization. Targeted B-Raf inhibitors such as vemurafenib and dabrafenib have revolutionized treatment of B-Raf V600E-mutant melanoma.
The AlphaLISA SureFire Ultra Human Total B-Raf V600E is a sandwich immunoassay for the quantitative detection of total B-Raf V600E 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.
PROTAC-mediated degradation of B-Raf V600E
A375 and HT-29 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). B-Raf V600E and Raf-1 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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 and HT-29 cells. As expected, treatment with V600E Degrader-1 resulted in a dose-dependent decrease of B-Raf V600E mutant levels while Raf-1 levels remained unchanged.
PROTAC-mediated degradation of B-Raf V600E
A375 and HT-29 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of SJF 0628 PROTAC for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). B-Raf V600E and Raf-1 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, treatment with SJF 0628 resulted in a dose-dependent decrease of B-Raf V600E mutant levels with no changes to Raf-1 levels.
B-Raf mutant vs wildtype differential expression
Adherent cells were grown to confluency in T175 flasks at 37°C, 5% CO2 and lysed with Lysis Buffer at a density of 0.5 x 106 cells/mL.
B-Raf V600E and B-Raf Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate 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.
B-Raf V600E is only detected in cancer cell lines that harbour this mutation, i.e. A375, SK-MEL-28 and HT 29 cells. As expected, no expression of B-Raf V600E was detected in B-Raf WT expressing cell lines like SW 48.
Assay sensitivity - cell lysate
Cell lysate was prepared from A375 cultured to confluency in a T175 flask and lysed in 6 mL of Lysis Buffer.
Lysate was serially diluted in Lysis Buffer and B-Raf V600E levels were evaluated using the AlphaLISA SureFire Ultra kit. 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 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. The assay can detect B-Raf B600E down to approximately 400 cells.
