Description
Overview
The Alpha
SureFire Ultra
Multiplex p-EGFR + Total EGFR assay kit is used to measure both phosphorylated and total levels of endogenous EGFR 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 EGFR analysis, and the 545 nm (Tb) signal corresponds to the total EGFR 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.
How it works
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 Capt
Sure
™ antibody, which binds the Capt
Sure
-tagged anti-phospho target antibody.
The Alpha 545 acceptor bead is coated with the Capt
Sure2
antibody, which binds the Capt
Sure2
-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.
