Description
The AlphaLISA SureFire Ultra total SMAD3 assay is a sandwich immunoassay for quantitative detection of SMAD3 (phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology.
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 reagents to run 500 wells in 384-well format, using a 20 μL reaction volume. The 10,000 point kit contains reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume. The 50,000 point kit contains reagents to run 500 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, which is biotinylated. Acceptor Beads are coated with a proprietary CaptSureTM 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
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.
