To 50 mL of conjugation buffer (100 mM Tris, pH 7.4, 5 mM MgCl 2 , 20 mM DTT, 40 mM ATP) was added ISG15 (Boston Biochem, 9.4 mM final) or GST-SUMO1 (Boston Biochem, 10.4 mM final) and biotinylated peptide 7-mer (biotin-VKAKIQD-OH, 250 mM final). The solution was mixed thoroughly and to this was added ISG15 activating enzyme (Boston Biochem, ISG15 E1, 50 nM final) and UbcH8 (Boston Biochem, 250 nM final), or SUMO activating enzyme (SAE1/SAE2 heterodimer, 50 nM final) and UbcH9 (250 nM final). The solution was mixed and incubated for 15 hours at 37uC. The reaction mixture was transferred to a microcentrifuge membrane filter (Vivascience, 5000 Da MWCO), diluted to 600 mL total volume with 50 mM Tris, pH 7.4 and concentrated at 4uC to 50 mL. This dilution/concentration procedure was repeated six times. The products were transferred to a clean tube and diluted with 50 mM Tris, pH 7.4 to a final volume of 100 mL. The SUMO1-and ISG15-linked biotinylated isopeptide was detected after transfer to a PVDF membrane (Perkin Elmer) with Streptavidin-HRP (Amersham).