AlphaLISA SureFire Ultra Human Phospho-BCKDHA (Ser292) Detection Kit, 10,000 Assay Points

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 permits the cells 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 AlphaLISA SureFire Ultra quality.

Inhibition of BCKDHA Phospho (Ser292) in HeLa cells

HeLa cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of BT2 for 8 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Ser292), Total BCKDHA and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 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.

As expected, treatment with BT2 resulted in a dose-dependent decrease in Phospho (Ser292) BCKDHA levels and a modest decrease in Total BCKDHA levels. Cofilin Total levels were unchanged (data not shown).

Knockout validation of BCKDHA Phospho (Ser292) assay

BCKDHA Phospho (Ser292) levels were assessed in HEK293T cells (WT) and BCKDHA KO (Abcam, ab266439) cell lines cultured to confluence in T175 flasks at 37°C, 5% CO₂.

Each flask was lysed in Lysis Buffer for 10 minutes at RT with shaking and BCKDHA Phospho (Ser292) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell 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 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.

BCKDHA Phospho (Ser292) was detected in WT but not KO cells. This confirms the specificity of the assay for the detection of BCKDHA protein.

BCKDHA assay sensitivity - cell lysate dilution

Cell lysate was prepared from HeLa cells cultured to confluence in a T175 flask and lysed in 4 mL of Lysis Buffer.

Lysate was serially diluted in Lysis Buffer and BCKDHA Phospho (Ser292) and Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL 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.

Approximate number of cells is indicated. The dotted line represents assay background. The BCKDHA assay has a broad dynamic range and can detect Phospho (Ser292) BCKDHA in less than 250 cells/datapoint.