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Fluorescent labeling of a protein enables the study of protein\u2010protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy\u2010to\u2010apply protocol for high\u2010stoichiometric, site\u2010specific labeling of a protein at its N\u2010terminus with <jats:italic>N<\/jats:italic>\u2010hydroxysuccinimide (NHS) esters as a means to measure protein\u2010protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent\u2010labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2\u2010E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. \u00a9 2021 Wiley Periodicals LLC.<\/jats:p><jats:p>This article was corrected on 18 July 2022. See the end of the full text for details.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Labeling a protein of interest at its N\u2010terminus with NHS esters through stepwise reaction<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Labeling a protein of interest at its N\u2010terminus with NHS esters through a one\u2010pot reaction<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Purifying the N\u2010terminal fluorescent\u2010labeled protein and determining its concentration and labeling efficiency<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Using MST to determine the binding affinity of an N\u2010terminal fluorescent\u2010labeled protein to a binding partner.<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay<\/jats:p>","DOI":"10.1002\/cpz1.14","type":"journal-article","created":{"date-parts":[[2021,1,21]],"date-time":"2021-01-21T20:17:42Z","timestamp":1611260262000},"source":"Crossref","is-referenced-by-count":22,"title":["N\u2010Terminal Protein Labeling with <i>N<\/i>\u2010Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein\u2010Protein Interactions Using Labeled Protein"],"prefix":"10.1002","volume":"1","author":[{"given":"Hanjie","family":"Jiang","sequence":"first","affiliation":[{"name":"Division of Genetics, Brigham and Women's Hospital, Department of Medicine and Biological Chemistry and Molecular Pharmacology Harvard Medical School  Boston Massachusetts"},{"name":"Department of Pharmacology and Molecular Sciences Johns Hopkins School of Medicine  Baltimore Maryland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip A.","family":"Cole","sequence":"additional","affiliation":[{"name":"Division of Genetics, Brigham and Women's 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