Label-Free Single-Molecule Pulldown for the Detection of Released Cellular Protein Complexes

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 mm(2). 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.

Automated summary

This article presents a label-free single-molecule pulldown (LFSMP) technique that allows imaging of intracellular proteins and complexes with single-molecule sensitivity and low sample consumption. The technique is based on plasmonic scattering imaging, which enables direct imaging of captured molecules without labels and quantification of binding kinetics. The authors demonstrate the detection principle of LFSMP, study the phosphorylation of protein complexes involved in a signaling pathway, and investigate the use of kinetic analysis to improve pulldown specificity.