Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy.

ABSTRACT: Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and th

SUBMITTER: Pisapati AV 

PROVIDER: S-EPMC7205595 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature