Labeling a protein with fluorophores using NHS ester derivitization

  • Methods Enzymol. 2014;536:87-94. doi: 10.1016/B978-0-12-420070-8.00008-8.
Jagpreet S Nanda  1 ,  Jon R Lorsch  2
Affiliations
  • 1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • 2. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: [email protected].
PMID: 24423269 DOI: 10.1016/B978-0-12-420070-8.00008-8
Abstract

N-hydroxysuccinimde (NHS) ester-mediated derivitization involves the reaction of this amine-reactive group with the primary amines of a protein or a biomolecule. Using NHS chemistry allows one to conjugate various fluorescent probes, biotin, and cross-linkers to primary amines. For example, we use NHS ester chemistry to fluorescently label the amino terminus of a protein with the dye, 5-(and-6)-carboxyfluorescein, succinimidyl ester (5(6)-FAM, SE).

Keywords
Efficiency of labeling calculation; Fluorescent labeling of protein; Fluorophores; Lyophilized protein; NHS ester derivitization; UV spectrophotometer.
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