Description
Overview Ataxia Telangiectasia and Rad3-related (ATR) is a serine/threonine protein kinase that serves as a master regulator of the DNA damage response, particularly in response to replication stress. ATR is activated by RPA-coated single-stranded DNA, leading to phosphorylation of downstream effectors including CHK1, p53, and H2AX. Upon activation, ATR initiates cell cycle checkpoints, stabilizes stalled replication forks, and promotes DNA repair. Hypomorphic mutations in ATR cause Seckel syndrome, while cancer cells often become dependent on ATR for survival due to elevated replication stress. ATR inhibitors are being developed as cancer therapeutics, showing synthetic lethality with ATM loss or BRCA mutations. The AlphaLISA SureFire Ultra Human and Mouse Total ATR is a sandwich immunoassay for the quantitative detection of total ATR 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 How it works Total-AlphaLISA SureFire Ultra assay principle The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate. The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted 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 targeted 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 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 permits the cells 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 AlphaLISA SureFire Ultra quality. Assay versatility ATR expression in various cell lines Adherent cells were grown to confluency in a T175 flask in complete medium and were lysed with Lysis Buffer at a density of 1 x 106 cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at a density of 3.2 x 106 cells/mL. ATR Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (10,000 adherent and 32,000 suspension cells) were 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. ATR expression was detected in a wide range of human and mouse cell lines. Assay sensitivity ATR assay sensitivity - cell lysate dilution Cell lysate was prepared from HeLa cultured to confluency in T175 flasks at 37°C, 5% CO2 . Each flask was treated with etoposide at 2 µM for 18 hours and lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysate was serially diluted in Lysis Buffer and ATR Phospho (Thr1989) 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 per datapoint is indicated. The dotted line represents assay background. This assay can detect ATR expression in less than 1,000 cells/datapoint.
