AlphaLISA SureFire Ultra Multiplex Human and Mouse Phospho-NF-κB (Ser536 & Total) Detection Kit, 100 Assay Points

Multiplex SureFire Ultra assay principle

The Multiplex SureFire Ultra assay simultaneously measures phospho and total protein at specific levels. 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 the cells 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 enables miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.