Branched-Chain Ketoacid Dehydrogenase E1-Alpha (BCKDHA) is the catalytic subunit of the BCKDC complex, which catalyzes the irreversible decarboxylation of branched-chain ketoacids derived from leucine, isoleucine, and valine. BCKDHA represents the rate-limiting step in branched-chain amino acid catabolism and is predominantly expressed in liver, muscle, kidney, and brain. Mutations in BCKDHA cause maple syrup urine disease (MSUD), characterized by accumulation of toxic ketoacid derivatives leading to neurological damage. MSUD severity varies depending on residual enzyme activity, ranging from classic severe forms to intermediate variants. Altered BCKDHA expression has been implicated in cancer metabolism, diabetes, and cardiovascular disease.
The AlphaLISA SureFire Ultra Human Phospho-BCKDHA (Ser292) is a sandwich immunoassay for the quantitative detection of phospho-BCKDHA (Ser292) 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
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 phosphorylated 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 phosphoprotein present in the sample.
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 enables cell 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 robust 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.
