AlphaLISA SureFire Ultra Human and Mouse Total B-Raf Detection Kit, 100 Assay Points

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 permits the cells 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 AlphaLISA SureFire Ultra quality.

Specific degradation of B-Raf using V600E Degrader-1

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 PROTAC 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.

Specificity of B-Raf Total assay

Total B-Raf protein 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 Total B-Raf using the AlphaLISA SureFire Ultra assay kit.

For the detection step, 10 µL of 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.

As expected, B-Raf was detected only in HeLa WT cells, demonstrating assay selectivity.

Expression of B-Raf in various cell lines

Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

Total B-Raf levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, suspension cell lines were further diluted 1:10 in Lysis Buffer, then 10 µL (approximately 4,000 cells) of cell lysate 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.

Total B-Raf protein is expressed in a wide range of cell types, with particularly high expression in MOLT-4, Ramos and mouse cell line NIH/3T3.