We recently described a novel fluorescent compound, 2-amino,6-amidobiotinyl-pyridine (BAP), that allows the tagging of oligosaccharides, their fractionation by reversed-phase HPLC with picomole scale detection, and the formation of functional neoglycoprotein equivalents with (strept) avidin for the detection of receptors and the generation of monospecific antibodies (Rothenberg et al., Proc. Natl Acad. Sci. USA, 90, 11939-11943, 1993). Here, we describe the enhancement of this approach by the following. (i) A simple one-step purification of BAP from its synthetic precursors and other reactants. (ii) Development of HPLC sizing column methods to quickly purify BAP-coupled oligosaccharides away from free BAP and other reactants. (iii) Development of anion-exchange and amine-adsorption HPLC procedures for the fractionation of BAP-oligosaccharide adducts by charge and size, respectively. (iv) Investigation of the affinity of BAP-oligosaccharides for (strept)avidin, confirming the formation of stable complexes. (v) The use of BAP for sensitive monosaccharide compositional analysis of glycoproteins. (vi) Formation of stable BAP adducts without reduction and its implications for the mechanism of adduct formation. These advances make available a multitude of techniques for the fractionation of BAP-coupled oligosaccharides based on several different physical parameters. Distinct species of BAP-coupled oligosaccharides can be isolated and subjected to detailed structural analysis. Such defined molecules form stable complexes with streptavidin that are effectively neoglycoproteins, which can be used in a variety of biological applications. Notably, all of these approaches require relatively inexpensive materials and conventional equipment available in most laboratories.