Parallel protein detection by solid-phase proximity ligation assay with real-time PCR or sequencing

Abstract

Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA-modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid-phase proximity ligation assays for soluble proteins using either real-time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed.

Keywords: NGS; PLA probes; PTM; SP-PLA; qPCR.