Magnetic Tweezers Protein Immobilization

Magnetic tweezers measurements were performed in fluid chambers that were functionalized by (3-aminopropyl)-trimethoxysilane in ethanol (1% v/v, Sigma-Aldrich, St. Louis, MO, USA), and glutaraldehyde (1% v/v, Sigma Aldrich) as described by Popa et al. [17 (link)]. HaloTag amine (O4) ligand (Promega, Madison, WI, USA) was added to create an attachment point for the protein of interest. Halo-(I83)₆ protein was diluted in 20 mM HEPES buffer and added to a functionalized flow cell. Chambers were washed with TRIS buffer and then streptavidin coated paramagnetic beads (Dynabeads M-270, Invitrogen, Waltham, MA, USA) were added to attach the biotinylated C-terminus of the protein.