Immunoblot of hISG15 fusion protein. Anti-hISG15 antibody, generated by immunization with recombinant human ISG15, was tested by immunoblot against a hISG15-GFP fusion protein produced in E.coli cell lysate soluble fraction. Dilution of the antibody between 1:200 and 1:1,000 showed strong reactivity specifically with hISG15 and ISG15 coupled proteins. Free hISG15 is indicated by the arrowhead. In this blot the antibody was used at a 1:200 dilution incubated overnight at 4°C in 5% non-fat dry milk in TTBS. Detection occurred using a 1:2000 dilution of HRPlabeled Donkey anti-Rabbit IgG for 1 hour at room temperature. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M.Malakhov, personal