Western blot using the anti-NAG-1 monoclonal antibody. The blot shows detection of recombinant NAG-1 protein present in Pichia pastoris whole cell lysates: lane 1 - yeast cell lysate expressing NAG-1 H variant with SUMO expression tag, lane 2 - yeast cell lysate expressing NAG-1 D variant with SUMO expression tag, lane 3 - yeast cell lysate expressing NAG-1 H variant and lane 4 - yeast cell lysate expressing NAG-1 D variant. Recombinant NAG-1 proteins correspond to 32 kDa and 16 kDa bands as indicated by the arrowheads. All lysates were run under reducing conditions. Primary antibody was used at a 1:1,000 dilution in TBS containg 1% BSA and 0.2% Tween, and reacted overnight at 4°C. Detection occurred using a 1:40,000 dilution of peroxidase conjugated Gt-a-Mouse IgG secondary antibody 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. Other detection systems will yield similar results.