AFM volumetric methods for the characterization of proteins and nucleic acids

Maria Eugenia Fuentes-Perez, Mark S Dillingham, Fernando Moreno-Herrero

Research output: Contribution to journalArticle (Academic Journal)

27 Citations (Scopus)

Abstract

The atomic force microscope overestimates lateral dimensions and underestimates heights of nanometer size objects such as proteins and nucleic acids. This has made researchers cautious of AFM measurements, even though there is no other technique capable of measuring topography with sub-nanometer precision. Nevertheless, several approaches for determining the stoichiometry of protein and protein-DNA complexes have been developed which show that, although the absolute values may be incorrect, the AFM volume is essentially proportional to the mass. This has allowed the determination of the mass of protein complexes with the help of a calibration curve. Here we review the main techniques for AFM volume measurements and detail a methodology that significantly reduces the associated errors. This method uses a fragment of DNA as a fiducial marker by which the volume of a protein is normalized. The use of fiducial markers co-adsorbed together with the protein of interest minimizes the contribution of tip-induced artifacts as they affect both the object of interest and the marker. Finally, we apply this method to the measurement of the length of single-stranded DNA. A linear relationship between length and volume was obtained, opening the door to studies of ssDNA intermediates formed during complex DNA transactions such as replication, recombination and repair.
Original languageEnglish
JournalMethods
DOIs
Publication statusPublished - 2013

Bibliographical note

Copyright © 2013. Published by Elsevier Inc.

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  • Activities

    • 1 - Research and Teaching at External Organisation

    Research collaboration with Moreno-Herrero Lab - Single molecule techniques.

    Mark S Dillingham (Contributor)

    2006 → …

    Activity: Other activity types - Research and Teaching at External Organisation

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