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

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

Author information

1
CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, FL, United States of America.

Abstract

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.

PMID:
30769341
DOI:
10.1088/1478-3975/ab0792
[Indexed for MEDLINE]

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