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The In Vitro Biotinylation Kit (with BirA Tagless Enzyme) is a kit designed for in vitro addition of biotin to biotin-acceptor peptides using BirA. This kit comes in a convenient, easy-to-use format and contains all the components necessary to label biotin-acceptor peptides with biotin. The BirA protein provided is a tagless recombinant biotin protein ligase (EC6.3.4.15). It efficiently catalyzes the specific attachment of biotin to the AviTag™. It is a robust reaction in a wide range of conditions, such as 20-40 µM of protein substrate (biotin-acceptor peptide) concentrations and can tolerate up to 1M NaCl and 20% glycerol in the reaction.