Lysozyme has been used extensively as a substrate to study the ubiquitin-dependent proteolytic pathway in vitro. This protein is an N-end rule substrate, since the recognition of protein requires the binding of an E3 ligase to the N-terminal lysine residue. Ubiquitin-lysozyme conjugates can be formed in the presence of ATP, ubiquitin and the appropriate conjugation enzyme source. This substrate protein can be used as a positive control with crude fractions and conjugation kits. Additionally, this lysozyme is labeled with biotin for the sensitive detection with avidin affinity reagents, but has all available lysines present for the efficient conjugation to ubiquitin.