IRDye NHS esters react with unblocked amines in peptides. NHS ester reactions usually proceed quickly and cleanly in either organic or aqueous solvents. While NHS esters continue to be widely used tools for biomolecule modification, their application to large peptides may be complicated by factors such as steric hindrance, amine inactivation by salt-bridging, and multiple labeling sites.Although a properly designed peptide can be labeled site-specifically by NHS ester chemistry, complex designs may incur cost-prohibitive materials and labor.To address the limitations of NHS ester chemistry, orthogonal technologies such as IRDye Maleimides and Click Chemistry Reagents have been developed to label non-amine functional groups. Orthogonal chemistry can also facilitate dual- or multi-dye labeling. As with NHS ester chemistry, optimal non-amine labeling is usually achieved by aqueous solution-phase reactions.Developed for: Odyssey® Classic, Odyssey CLx, Odyssey Sa, and Aerius Imaging Systems