Description
BCR-ABL1 is an oncogenic fusion protein resulting from the Philadelphia chromosome translocation t(9;22), which joins the BCR gene on chromosome 22 to the ABL1 tyrosine kinase gene on chromosome 9. The fusion protein exhibits constitutive tyrosine kinase activity independent of normal regulatory mechanisms, driving uncontrolled cell proliferation through activation of RAS/MAPK, PI3K/AKT, and JAK/STAT pathways. BCR-ABL1 is the causative driver in chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemias (ALL). The kinase domain of BCR-ABL1 is the target of tyrosine kinase inhibitors including imatinib, dasatinib, and nilotinib, which have revolutionized CML treatment and transformed it from a fatal disease to a manageable chronic condition. Resistance mutations in the kinase domain, particularly T315I, have driven development of next-generation inhibitors such as ponatinib.
The AlphaLISA SureFire Ultra Human Phospho-BCR-ABL1 Tyr245 Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-BCR-ABL1 Tyr245 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.
Tyrosine kinase inhibitors decrease BCR-ABL1 phosphorylation in K-562 cells
K-562 cells were seeded in a 96-well plate (100,000 cells/well) in complete medium and treated with increasing concentrations of Imatinib, Nilotinib or Asciminib for 6 hours at 37°C, 5% CO2.
After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BCR-ABL1 Phospho (Tyr412), Phospho (Tyr245) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 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.
As expected, treatment with tyrosine kinase inhibitors resulted in a dose-dependent decrease of Phospho (Tyr412) and Phospho (Tyr245) BCR-ABL1 levels, while Total levels remained unchanged.
Assay sensitivity - cell lysate
Cell lysate was prepared from K-562 cells prepared at 4 x 106 cells/mL in HBSS. Cells were lysed with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and BCR-ABL1 Phospho (Tyr412), Phospho (Tyr245) and Total 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 BCR-ABL1 Phospho (Tyr245) levels in less than 400 cells.
