Protocol to identify DNA-binding proteins recognizing nucleotide repeat dsDNAs

Zhiyuan Huang; Yixin Zhou; Yang Liu; Jiou Wang

doi:10.1016/j.xpro.2024.103013

Protocol to identify DNA-binding proteins recognizing nucleotide repeat dsDNAs

STAR Protoc. 2024 Apr 11;5(2):103013. doi: 10.1016/j.xpro.2024.103013. Online ahead of print.

Authors

Zhiyuan Huang¹, Yixin Zhou¹, Yang Liu¹, Jiou Wang²

Affiliations

¹ Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
² Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address: jiouw@jhu.edu.

Abstract

DNA-binding proteins perform diverse functions, including regulating cellular growth and orchestrating chromatin architecture. Here, we present a protocol to discover proteins specifically interacting with a hexanucleotide repeat DNA, the expansion of which is known as the most frequent genetic cause of familial C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. We describe steps to fish out DNA-binding proteins recognizing double-stranded repeat DNAs using a SILAC (stable isotope labelling by amino acids in cell culture)-based approach and validate the results using electrophoretic mobility shift assay. For complete details on the use and execution of this protocol, please refer to Liu et al.¹.

Keywords: Cell Biology; Cell culture; Cell separation/fractionation; Cell-based Assays; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification.