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 (F-340, F-360, F-370, F-372) and conjugation kits (K-915, K-930, K-935, K-960). 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.