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Binding Affinity Quantifications of the Bacteriophage Mu DNA Modification Protein Mom Using Microscale Thermophoresis (MST)

Udupa, S and Nagaraja, V and Karambelkar, S (2022) Binding Affinity Quantifications of the Bacteriophage Mu DNA Modification Protein Mom Using Microscale Thermophoresis (MST). In: Bio-protocol, 12 (14).

Full text not available from this repository.
Official URL: https://doi.org/10.21769/BioProtoc.4472


Epigenetic modifications play diverse roles in biological systems. Nucleic acid modifications control gene expression, protein synthesis, and sensitivity to nucleic acid-cleaving enzymes. However, the mechanisms underlying the biosynthesis of nucleic acid modifications can be challenging to identify. Studying protein-ligand interactions helps decipher biosynthetic and regulatory pathways underlying biological reactions. Here, we describe a fluorescence labeling-based quantitative method for unraveling the biomolecular interactions of bacteriophage Mu DNA modification protein Mom with its ligands, using microscale thermophoresis (MST). Compared to traditional methods for studying protein-biomolecular interactions, MST requires significantly lower sample amounts, volumes, and analysis time, thus allowing screening of a large number of candidates for interaction with a protein of interest. Another distinguishing feature of the method is that it obviates the need for protein purification, often a time- and resource-consuming step, and works well with whole or partially purified cell extracts. Importantly, the method is sensitive over a broad range of molecular affinities while offering great specificity and can be used to interrogate ligands ranging from metal ions to macromolecules. Although we established this method for a DNA modification protein, it can easily be adapted to study a variety of molecular interactions engaged by proteins.

Item Type: Journal Article
Publication: Bio-protocol
Publisher: Bio-protocol LLC
Additional Information: The copyright for this article belongs to the Bio-protocol LLC.
Keywords: Acetyl coenzyme A (acetyl CoA); Anti-restriction; Bacteriophage Mu; DNA modification; Fe2 binding; GNAT fold; Kd-dissociation constant; Microscale thermophoresis (MST); mom
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited: 23 Aug 2022 05:13
Last Modified: 23 Aug 2022 05:13
URI: https://eprints.iisc.ac.in/id/eprint/76154

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