Anti-Human SUMO-3 Antibody - Western Blot. Western blot analysis is shown using Affinity Purified anti-Human SUMO-3 antibody to detect GFP-SUMO fusion proteins (arrowheads). Panel A. Increasing concentrations of human GFP-SUMO-3 were run on a SDS-PAGE, transferred onto nitrocellulose, and blocked for 1 hour with 5% non-fat dry milk in TTBS, and probed overnight at 4°C with a 1:1000 dilution of anti-hSUMO-3 antibody in 5% non-fat dry milk in TTBS. Detection occurred using a 1:1000 dilution of HRP-labeled Donkey anti-Rabbit IgG for 1 hour at room temperature. A chemiluminescence system was used for signal detection (Roche). Panel B. Specificity of the antibody was confirmed by SDS-PAGE of 5 ug of various GFP-SUMO constructs followed by transfer onto nitrocellulose. Lanes: 1. MW marker, 2. GFP-human SUMO-3, 3. GFP-human SUMO-1, 4. GFP-yeast SUMO, 5. GFP-Arabidopsis thaliana, SUMO-1, 6. GFP- Arabidopsis thaliana SUMO-2, 7. GFP-tomato SUMO. After blocking for 1 hour with 5% non-fat dry milk in TTBS, the blot was probed overnight at 4°C with anti-hSUMO-3 antibody diluted and detected as above. Only the human GFP-SUMO-3 band was visualized by chemiluminescence, and no cross-reactivity with other SUMO family members was observed.