Western blot shows detection of recombinant NAG-1 protein (arrow) present in Pichia pastoris whole cell lysates: lane 1 - yeast cell lysate expressing NAG-1 H variant with SUMO expression tag at 36 kDa; lane 2 - yeast cell lysate expressing NAG-1 D variant with SUMO expression tag at 36 kDa; lane 3 - yeast cell lysate expressing NAG-1 H variant; and lane 4 - yeast cell lysate expressing NAG-1 D variant. All lysates were run under reducing conditions. Primary antibody was used at a 1:1000 dilution in TBS containing 1% BSA and 0.2% Tween, and reacted overnight at 4°C. For detection, a 1:40,000 dilution of peroxidase conjugated Gt-a-Mouse IgG secondary antibody was used in Blocking Buffer for Fluorescent Western Blotting (MB-070) for 30 min at room temperature. Molecular weight estimation was made by comparison to prestained MW markers. Image was captured using the BioRad Versadoc 4000MP Imaging System.