[14] Protein biotinylation

This chapter discusses the procedures by which researchers have incorporated biotin moieties into proteins. Several laboratories have reported the use of an avidin-based assay for detecting the extent of biotinylation. An effective method for detecting the average number of biotin groups per protein molecule would be to first remove biotin from the protein and then to determine the amount of biotin in the sample. The percentage of biotinylated peanut agglutinin (PNA) molecules that are accessible for binding avidin in a given sample is assessed by quantitative affinity chromatography on an avidin column. An easy and effective way to determine nonspecific interactions of the biotinylated lectin is to use dot blots. A variety of target materials can be employed for this purpose. Because PNA recognizes the exposed penultimate galactose residues in desialylated sialoglycoconjugates, asialofetuin is an excellent positive control for this assay. Altering the relative concentration or ratio of reagent and/or antibody alters the extent or efficiency of biotinylation.