Preparation of SNAPf-Beads for Colocalization Single-Molecule Spectroscopy (CoSMoS) of RNA-Protein Complexes

Methods Mol Biol. 2020:2113:17-22. doi: 10.1007/978-1-0716-0278-2_2.

Abstract

The SNAPf-tag is a chemical tag that allows rapid and highly specific covalent labeling of proteins even in the full complexity of the cellular environment. The SNAPf-tag has been instrumental to study native RNA-protein complexes at single-molecule resolution in their cellular environment as efficient labeling of the RNAs and proteins of interest is essential for this colocalization single-molecule spectroscopy (CoSMoS) technique. However, removal of excessive benzylguanine dye after the labeling reaction has remained challenging. Here, we describe a strategy to remove excessive benzylguanine dye using SNAPf-tag coated beads as sponges.

Keywords: CoSMoS; Colocalization single-molecule spectroscopy; Protein labeling; RNA spectroscopy; RNA-protein complex; RNP; Single-molecule fluorescence microscopy; Single-molecule spectroscopy; TIRF microscopy.

Publication types

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

MeSH terms

  • Fluorescent Dyes / chemistry
  • Microscopy, Fluorescence
  • Molecular Imaging
  • RNA / chemistry
  • RNA / metabolism*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*
  • Single Molecule Imaging / methods*
  • Staining and Labeling

Substances

  • Fluorescent Dyes
  • RNA-Binding Proteins
  • RNA