The amine group is the most common target for modifying protein molecules because it is abundant in the majority of proteins either due to the presence of lysine bearing amino side chain functionality or the N-terminal-amine. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility: NHS-esters and sulfo-NHS-esters.

NHS-esters of biotin are water-insoluble and membrane-permeable because they do not possess a charged group

Sulfo-NHS-esters are water soluble and useful for applications that cannot tolerate organic solvents. Sulfo-NHS-esters of biotin are recommended for use as cell surface biotinylation reagents. Because of the charged sulfonate group, these compounds do not penetrate the plasma membrane, thus sulfo-NHS-esters biotinylation is restricted to the cell surface.

PEGylated esters transfer water solubility to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG spacer arm also gives the reagent a long and flexible connection to minimize steric hindrance for binding to avidin molecules.

They may be used for biotinylating internal as well as external cellular components. Since NHS-esters of biotin are water-insoluble, for reactions in aqueous solution they must first be dissolved in an organic solvent, such as DMSO and DMF.