This kit is for the formation of ubiquitinated substrate proteins. The enzymes supplied represent the full compliment of purified conjugation enzymes (E1, E2s, and E3s) that are found in Fraction II. Conjugation Fraction A contains predominantly E1 and E2 enzymes, while Conjugation Fraction B contains predominantly E3 and deubiquitinating enzymes. The enzymes have been tested and shown to work with typical [125I]-labeled substrate proteins such as lysozyme and β- lactoglobulin. This conjugation fraction also contains ubiquitin C-terminal hydrolases and therefore the addition of Ubiquitin Aldehyde (U-201) is recommended for the inhibition of UCHs to improve overall conjugate yield. The supplied fractions do not contain 20S or 26S proteasomes or any other protein degradation activity. If the substrate being conjugated requires E2 or E3 enzymes not found in Fraction II, the reaction can be supplemented with enzymes from Fraction I (F-375).

NOTE: Kit contains reagents sufficient for 6 x 100 μl reactions.