The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules.

ABSTRACT: Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with ³² P-labeled DNA quantify K_d values from 10^-12 to 10^-8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio k_off /k_on determines K_d . However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibriu

SUBMITTER: Weeramange CJ 

PROVIDER: S-EPMC7096710 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature