Immunoblot of ySUMO fusion protein. Anti-ySUMO antibody, generated by immunization with recombinant yeast SUMO, was tested by immunoblot 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 IRDye (R) 800 Conjugated Affinity Purified Goat-anti-Mouset 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.