BCL-2 Associated X-protein (BAX) is a pro-apoptotic member of the BCL-2 family that mediates mitochondrial outer membrane permeabilization to initiate the intrinsic apoptotic pathway. Upon apoptotic stimulation, BH3-only proteins activate BAX, causing translocation to mitochondria, oligomerization, and pore formation. BAX oligomers release cytochrome c and other pro-apoptotic factors that activate caspases and execute cell death. Loss of BAX function contributes to cancer development, chemotherapy resistance, and autoimmune diseases through impaired apoptotic responses. Therapeutic strategies aim to restore BAX-mediated apoptosis through BH3 mimetics or direct BAX activators.
The AlphaLISA SureFire Ultra Human Total BAX is a sandwich immunoassay for the quantitative detection of total BAX 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
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes 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 target 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 protein present in the sample.
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.
Induction of Bax in etoposide treated cells
U2OS 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 etoposide for 24 hours in serum-free media.
After treatment, cells washed with HBSS and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total Bax and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,000 cells or 200 cells for Cofilin) 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, etoposide triggered a significant increase in the levels of Total Bax whilst Total Cofilin remained unchanged.
Knockout validation of Bax Total assay
Bax Total levels were assessed in HeLa Wild Type (WT) and Bax knockout (KO) cells (Abcam ab255363). Cells were seeded at various densities in a 96 well plate in complete medium and incubated overnight at 37°C, 5% CO2.
The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Bax 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.
Bax Total signal was only detected in the WT cells confirming the specificity of the assay.
Expression of Bax in various human cell lines
Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 200 µL of Lysis Buffer.
Suspension cells were harvested, washed with HBSS and lysed at various cell densities. Cell lysates were diluted to 1 x 106 cells/mL with Lysis Buffer.
Bax Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells or 10,000 suspension 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.
Bax expression was detected in a wide range of human adherent and suspension cell lines.
Assay sensitivity - cell lysate dilution
Cell lysate was prepared from A549 cells cultured to confluency in a T175 flask and lysed in 16 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and Total Bax levels were evaluated by AlphaLISA SureFire Ultra. 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 Total Bax down to 50 cells/datapoint.
