Protocol for the production and purification of an i-Motif-specific nanobody

Mahdi Zeraati; Samuel E Ross; Behnaz Aghaei; Alvaro González Rajal; Cecile King; Marcel E Dinger

doi:10.1016/j.xpro.2023.102729

Protocol for the production and purification of an i-Motif-specific nanobody

STAR Protoc. 2023 Dec 15;4(4):102729. doi: 10.1016/j.xpro.2023.102729. Epub 2023 Nov 22.

Authors

Mahdi Zeraati¹, Samuel E Ross², Behnaz Aghaei³, Alvaro González Rajal³, Cecile King³, Marcel E Dinger⁴

Affiliations

¹ School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address: mahdi.zeraati@sydney.edu.au.
² School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW 2006, Australia.
³ School of Biotechnology and Biomolecular Sciences, Faculty of Science, The University of New South Wales Sydney, Sydney, NSW 2052, Australia.
⁴ School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address: marcel.dinger@sydney.edu.au.

Abstract

Intercalated motifs or i-Motifs (iMs) are nucleic acid structures formed by cytosine-rich sequences, which may regulate cellular processes and have broad applications in nanotechnology due to their pH-dependent nature. We have developed an iM-specific nanobody (iMbody) that can recognize iM DNA structures regardless of their sequences, making it a versatile research tool for studying iMs in various contexts. Here, we provide a protocol for the bacterial expression and His-tag purification of iMbody. We then describe procedures for performing ELISA and immunostaining using iMbody.

Keywords: Antibody; Biotechnology and bioengineering; Genomics; Microscopy; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification.

MeSH terms

DNA* / metabolism
Nanotechnology* / methods
Nucleotide Motifs

Substances

DNA