A flexible, robust microbead-based assay for quantification and normalization of target protein concentrations

Abstract

Although there are many methods for quantifying the concentration of specific proteins in samples, current techniques are technically challenging or do not easily lend themselves to normalization. Here, we describe a microbead-based assay for quantifying specific protein concentration(s) that is high-throughput, inexpensive, simple-to-use, and intrinsically incorporates normalization against the sample total protein content. This assay, termed the FRANC assay, exploits high affinity biotin-streptavidin binding to couple sample proteins to streptavidin-labelled magnetic microbeads. Proteins are then antibody-probed, followed by labeling of proteins on the microbead with fluorescent dye, and flow cytometry-based analysis. The FRANC assay demonstrates detection limits for target proteins in the femtogram range, with a linear range up to as much as 10 ng. Normalization of target protein concentrations resulted in an 80% reduction in variability as compared to non-normalized measurements. We conclude that the FRANC assay offers attractive advantages over current methods for quantifying specific protein(s) in samples.

Keywords: Assay development; Biotin-streptavidin; Flow cytometry; Protein expression.