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.
In the AlphaLISA SureFire Ultra assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of protein present in the sample.
AlphaLISA SureFire Ultra™ kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
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.
Induction of phosphorylation in A431 EGF treated cells
A431 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 serum starved for 2 hours and then treated in serum free medium with increasing concentrations of EGF for 10 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1068) and Total EGFR 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, EGF induced a dose-dependent increase in Phospho (Tyr1068) EGFR levels while Total levels remained unchanged.
AG1478 inhibits EGFR phosphorylation in A431 cells
A431 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of AG1478 for 2 hours in serum free media. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1068) and Total EGFR levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, AG1478 decreased Phospho (Tyr1068) EGFR levels in a dose-dependent manner while Total levels remained unchanged.
Rociletinib inhibits EGFR phosphorylation in NCI-H1975 cells
NCI-H1975 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 Rociletinib for 24 hours. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1068) and Total EGFR 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, Rociletinib decreased Phospho (Tyr1068) EGFR levels in a dose-dependent manner while Total levels remained unchanged.
EGFR assay sensitivity - cell lysate dilution
Cell lysate was prepared from A431 cells cultured to confluence in T175 flasks in complete medium. Cells were treated with 2 µg/mL EGF for 10 minutes and then lysed in 8 mL of Lysis Buffer.
Lysates were serially diluted in Lysis Buffer and assayed for Phospho (Tyr1068) and Total EGFR levels using respective AlphaLISA SureFire Ultra kits. 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/datapoint is indicated. The dotted line represents assay background. The assay can detect EGFR protein down to 50 cells/datapoint.
