Facile single-molecule pull-down assay for analysis of endogenous proteins

Phys Biol. 2019 Mar 22;16(3):035002. doi: 10.1088/1478-3975/ab0792.


The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane-Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab')2 or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigen-Antibody Reactions
  • Humans
  • Immunoglobulin Fab Fragments / analysis*
  • Immunoglobulin G / analysis*
  • Immunoprecipitation
  • Microscopy, Fluorescence
  • Polysorbates / chemistry*
  • Silanes / chemistry*


  • Immunoglobulin Fab Fragments
  • Immunoglobulin G
  • Polysorbates
  • Silanes
  • dichlorodimethylsilane