FIG. 2.

Biotin-labeled ubiquitin is efficiently used by the ubiquitin conjugation system to form ubiquitin conjugates. A, 2 μg of wild-type ubiquitin (wt Ub) or purified Lys⁶-biotinylated ubiquitin (K6-Bio Ub) was added to fraction II of rabbit reticulocytes in the presence of 2 mM ATP in a 25-μl assay. After incubation at 37 °C for 30 min, the reaction was terminated by addition of 25 μl of 2× SDS gel loading buffer. The mixture was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The levels of ubiquitin conjugates (Ub-conj.) were detected by Western blot analysis using anti-ubiquitin antibodies. K6K48-dibio Ub, Lys⁶/Lys48-dibiotinylated ubiquitin; K6K63-dibio Ub, Lys⁶/Lys⁶³-dibiotinylated ubiquitin; K6K48K63-tribio Ub, Lys⁶/Lys⁴⁸/Lys⁶³-tribiotinylated ubiquitin. B, 0—4 μg of unmodified ubiquitin (lanes 1—6) or Lys⁶-biotinylated ubiquitin (lanes 7—12) was added to the conjugation assays, which contained 0.5 μgof ¹²⁵I-labeled wild-type ubiquitin. Ubiquitin conjugation was performed as described for A, and ubiquitin conjugates were monitored by autoradiography. C, the results in B were quantitated by densitometry. D, 0—4 μg of wild-type ubiquitin was added to the conjugation assays, which contained 0.5 μg of Lys⁶-biotinylated ubiquitin. Biotinylated ubiquitin conjugates were determined with horseradish peroxidase-conjugated avidin using a chemiluminescence detection kit.