Qian Gao, Joella Blas, and Efthalia Gerasimopoulos, Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA
Bio-Plex assays allow detection of mutiple target proteins in a small volume of sample. The assays are based on detection of signal from tagged fluorescent beads (xMAP technology) that have bound to each target protein. Typically, each type of bead is conjugated with distinct antibodies that recognize one of the target proteins. Appropriate sets of beads are then selected that enable simultaneous, or multiplex, assays of the proteins of interest in the sample. Bio-Plex phosphoprotein assays report the level of a protein that is phosphorylated at a specific site or sites, while total target assays report the level of the protein regardless of its phosphorylation state.
Typically, because of epitope binding competition for the same target protein, a total target assay should not be multiplexed with the corresponding phosphoprotein assay. Also, because the pair of antibodies used in the two assays differ, the signal from a total target assay may not be comparable to that of its corresponding phosphoprotein assay, so it is not appropriate to calculate the percentage of phosphorylated target protein using the results from a total and a phosphoprotein assay in the same reaction. However, because maximizing the number of assays in a particular reaction is desirable to conserve sample, in this note, we present results from a case study testing the feasibility of multiplexing a Bio-Plex total target assay with Bio-Plex phosphoprotein assays for different target proteins. This study was designed to answer two questions:
Can a total target assay be multip