The 26S proteasome is an ATP-dependent, multisubunit (~31), barrel-shaped molecular machine with an apparent molecular weight of ~2.5 MDa. It consists of a 20S proteolytic core complex which is crowned at one or both ends by 19S regulatory subunit complexes. The 19S regulatory subunits recognize ubiquitinated proteins and play an essential role in unfolding and translocating targets into the lumen of the 20S subunit. An enzymatic cascade is responsible for the attachment of multiple ubiquitin molecules to lysine residues of proteins targeted for degradation. Several genetic diseases are associated with defects in the ubiquitin-proteasome pathway. Some examples of affected proteins include those linked to cystic fibrosis, Angelmans syndrome, and Liddle syndrome.

EP-500 is the protein fraction of HeLa cell extract that does not bind to anion exchange resin. EP-500 contains ubiquitin, ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). EP-500 is ideal for demonstrating if FI enzymes are critical for degradation of in vitro substrates as well as in vitro conjugation activity.