Description
The retinoblastoma protein (Rb), encoded by RB1, is a tumor suppressor that regulates the cell cycle by inhibiting E2F transcription factors, thereby blocking S phase entry. Rb is inactivated via phosphorylation by cyclin-dependent kinases, enabling cell cycle progression. Mutations in RB1 cause retinoblastoma and are implicated in various cancers, including osteosarcoma and lung cancer, due to the loss of cell cycle control.
The AlphaLISA SureFire Biotin-Free Human and Mouse Phospho-Rb (Thr826) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-Rb 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 Biotin Free kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Biotin Free assay principle
The Phospho-AlphaLISA SureFire Biotin Free assay measures a protein target 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 a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with CaptSure3 tag. 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 Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Biotin Free 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 quality.
Decrease of Rb Phospho (Thr826) in Palbociclib treated cells
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of Palbociclib for 24 hours at 37°C, 5% CO2.
After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Phospho (Thr826) and Rb Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer,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, Palbociclib triggered a dose-dependent decrease in the levels of Phospho (Thr826) and Total Rb.
Decrease of Rb Phospho (Thr826) in CDK6 PROTAC treated cells
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of BSJ-03-123 (CDK6 PROTAC) for 24 hour at 37°C, 5% CO2.
After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Phospho (Thr826) and ERK1/2 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer, 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, BSJ-03-123 induced a significant decrease in the levels of Rb Phospho (Thr826) while Total ERK1/2 levels remain unchanged.
