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. 2021:2263:381-395.
doi: 10.1007/978-1-0716-1197-5_18.

Characterization of Protein-Nucleic Acid Complexes by Size-Exclusion Chromatography Coupled with Light Scattering, Absorbance, and Refractive Index Detectors

Ewa Folta-Stogniew  1
Affiliations

Characterization of Protein-Nucleic Acid Complexes by Size-Exclusion Chromatography Coupled with Light Scattering, Absorbance, and Refractive Index Detectors

Ewa Folta-Stogniew. Methods Mol Biol. 2021.

Abstract

Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS) enables determination of the molecular weight, oligomeric state, and stoichiometry of protein-nucleic acid complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a challenge in determination of molecular weight and stoichiometry based solely on the elution position from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the complex between the reverse transcriptase (RT) domain from group II intron-maturase from Eubacterium rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting repressor (FIR).

Keywords: Laser light scattering; Molecular weight; Oligomeric state; Protein–nucleic acid complex; SEC/MALS; Size-exclusion chromatography (SEC); Stoichiometry.

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