B-Raf Total Assay Kit Human and Mouse

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Data obtained with a 2-plate, 2-incubation protocol. A375 cells were seeded at 10K cells/well and incubated overnight. Cells were treated with a B-Raf PROTAC, B-Raf V600E Degrader-1, at the indicated concentrations for 24 hours. Cells were lysed with Lysis Buffer and assayed separately for Phospho (Ser446) and Total B-Raf and Total Raf-1 using respective SureFire® Ultra™ kits. Equivalent to approximately 1,000 cells/datapoint.

HeLa wild type (WT) and HeLa B-Raf knockout (KO) (Abcam) cells were cultured to confluency in T175 flasks. Each flask was lysed in 4 mL of Lysis Buffer, serially diluted in Lysis Buffer and evaluated for Total B-Raf levels using the SureFire® Ultra™ kit. Approximate number of cells/datapoint is indicated.

Data obtained from measurement of Total B-Raf protein levels in various cell types. Adherent cell lines were seeded at 40K cells/well and incubated overnight and lysed with Lysis Buffer. Suspension cell lines were seeded at 400K cells/well and lysed with Lysis Buffer. Suspension and adherent cell lysates were then assayed for Total B-Raf using the SureFire® Ultra™ kit. Equivalent to approximately 4,000 cells/datapoint for adherent and 40,000 cells/datapoint for suspension cell lines.