Description
The Alpha SureFire® Ultra™ Multiplex p-SMAD (Ser465/467) (Eu) + Total SMAD2 (Tb)assay kit is used to measure both phosphorylated and total levels of endogenous SMAD in cellular lysates in a multiplexing Alpha no-wash assay. No specially engineered cell lines are required. The 615 nm (Eu) signal corresponds to the phosphorylated SMAD2 analysis, and the 545 nm (Tb) signal corresponds to the total SMAD2 analysis. This kit has been formulated to provide superior signal:background assay windows and to perform without interference in the presence of extraneous antibodies, making it amenable to the study of therapeutic and blocking antibodies.
No-wash steps, no separation steps
Multiplexing assay
Sensitive detection
No interference from exogenous antibodies
Results in less than 3 hours
In this assay, the Alpha 615 Acceptor bead is coated with the CaptSure™ antibody, which binds the CaptSure-tagged anti-phospho target antibody. The Alpha 545 Acceptor bead is coated with the CaptSure2 agent, which binds the CaptSure2 tagged anti-total target protein antibody. The Alpha Donor bead binds the biotinylated anti-total target protein antibody.
In this assay, the Alpha 615 Acceptor bead is directly-conjugated with an antibody to the phosphorylated site on the target protein. The Alpha 545 Acceptor bead is coated with the CaptSure™ agent, which binds the CaptSure tagged anti-total target protein antibody. The Alpha Donor bead binds the biotinylated anti-total target protein antibody.
Multiplex SureFire Ultra assay principle
The Multiplex SureFire Ultra assay simultaneously measures phospho and total protein at a specific level in a biological sample (e.g. cell lysate). It utilizes three antibodies that recognize the phospho epitope and two distal epitopes on the target protein. The assay requires three bead types: two distinct Acceptor Beads and one Donor Bead.
The Alpha 615 Acceptor Bead is coated with the CaptSure™ antibody, which binds the CaptSure-tagged anti-phospho target antibody.
The Alpha 545 Acceptor Bead is coated with the CaptSure2 antibody, which binds the CaptSure2-tagged anti-total target protein antibody.
The Alpha Donor Bead binds the biotinylated anti-total target protein antibody.
When the phosphorylated protein is present, the three antibodies bring the Donor and Acceptor Beads close together, allowing singlet oxygen to transfer energy and excite both Acceptor Beads. This generates a luminescent Alpha signal at 615nm and 545nm. The 615nm (Eu) signal is directly proportional to the amount of phosphorylated protein, while the 545nm (Tb) signal corresponds to the total protein levels.
Multiplex 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 Multiplex 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 different targets.
Multiplex SureFire Ultra one-plate assay protocol
Detection of Phosphorylated/Total target proteins with Multiplex 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.
