In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis.

Stephanie C Ems-McClung, Claire E Walczak

Methods in molecular biology (Clifton, N.J.) 2020

filter terms:

Proper cell division and the equal segregation of genetic material are essential for life. Cell division is mediated by the mitotic spindle, which is composed of microtubules (MTs) and MT-associated proteins that help align and segregate the chromosomes. The localization and characterization of many spindle proteins have been greatly aided by using GFP-tagged proteins in vivo, but these tools typically do not allow for understanding how their activity is regulated biochemically. With the recent explosion of the pallet of GFP-derived fluorescent proteins, fluorescence-based biosensors are becoming useful tools for the quantitative analysis of protein activity and protein-protein interactions. Here, we describe solution-based Förster resonance energy transfer (FRET) and fluorescence assays that can be used to quantify protein-protein interactions and to characterize protein conformations of MT-associated proteins involved in mitosis.

Citation

Stephanie C Ems-McClung, Claire E Walczak. In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis. Methods in molecular biology (Clifton, N.J.). 2020;2101:93-122