Anti SUMO  

Western blot of ySUMO fusion protein. Anti-ySUMO antibody, generated by immunization with recombinant yeast SUMO, was tested by western blot against a SUMO-GFP fusion protein (lane 2). While the actual molecular weight of the fusion protein is 39 kDa, the protein migrates as a 49 kDa band (arrowhead). No reactivity is seen for lane 1 which contains His-tagged GFP protein. The membrane was blocked using BLOTTO. Primary antibody was used at a 1:1,000 dilution in BLOTTO. The membrane was washed and reacted with a 1:10,000 dilution of IRDyeR 800 Conjugated Affinity Purified Goat-anti-Mouse IgG (H&L) MX10 (800 nm channel). Molecular weight estimation was made by comparison to prestained MW markers indicated at the right (lane M, 700 nm channel). Other detection systems will yield similar results.

Most modifiers mature by proteolytic processing from inactive precursors (a; amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thiolesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein) /CBC(cullin-2/elongin B/elonginC)-like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin. Data contributed by S.Jentsch.