Abstract
Precise and sensitive detection of intracellular proteins and complexes is key to the understanding of signaling pathways and cell functions. Here, we present a label-free single-molecule pulldown (LFSMP) technique for the imaging of released cellular protein and protein complexes with single-molecule sensitivity and low sample consumption down to a few cells per mm2. LFSMP is based on plasmonic scattering imaging and thus can directly image the surface-captured molecules without labels and quantify the binding kinetics. In this paper, we demonstrate the detection principle for LFSMP, study the phosphorylation of protein complexes involved in a signaling pathway, and investigate how kinetic analysis can be used to improve the pulldown specificity. We wish our technique can contribute to uncovering the molecular mechanisms in cells with single-molecule resolution.