AlphaLISA SureFire Ultra Human Total A-RAF Detection Kit, 10,000 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 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.

Assay specificity - Knockout validation

A-Raf levels were assessed in HEK293T wild type (WT) and A-Raf KO (Abcam, ab266351) cell lines cultured to confluency in T175 flasks at 37°C, 5% CO₂.

Each flask was lysed in Lysis Buffer for 10 minutes at RT with shaking and A-Raf levels were evaluated using the AlphaLISA SureFireBiotin Free assay. For the detection step, 10 µL of lysate (10,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

A-Raf was detected in WT but not in the KO cells, confirming the specificity of the assay for the detection of A-Raf protein.

A-Raf expression in various cell lines

Various cell lines were seeded in a 96-well plate (40,000 cells) in complete medium and incubated for 24 hours at at 37°C, 5% CO₂.

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm) andA-Raf levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (4,000 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.

A-Raf expression was detected in a range of human cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from MCF7 cells cultured to confluence in a T175 flask and lysed with 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and A-Raf levels were evaluated using the AlphaLISA SureFire assay. 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 room temperature. 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect A-Raf expression in less than 500 cells/datapoint.