Ubiquitination mix

50 mg/mL creatine phosphokinase

1 mM DTT (dithiothreitol)

5 mM MgCl₂

1 pmol E1

5 pmol E2-C

1-2 pmol (~10⁵ cpm) of ¹²⁵I-labeled cyclin B (13-91)/protein A

10 mM phosphocreatine

1 mg/mL rcm-BSA (bovine serum albumin, reduced, carboxymethylated)

40 mM Tris-Cl (pH 7.6)

50 μM ubiquitin

1 μM ubiquitin aldehyde

Prepare fresh and keep at 4°C.

E1 was purified from human cells utilizing the method described in Hershko et al. (Hershko A, Heller H, Elias S, Ciechanover A. 1983. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem 258: 8206-8214).

Recombinant E2-C was expressed in bacteria as described in Aristarkhov et al. (Aristarkhov A, Eytan E, Moghe A, Admon A, Hershko A, Ruderman JV. 1996. E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins. Proc Natl Acad Sci 93: 4294-4299) and purified by gel filtration on a Superdex FPLC column.

Sea urchin cyclin B derivative cyclin B(13-91)/protein A was obtained and iodinated as described by Glotzer et al. (Glotzer M, Murray AW, Kirschner MW. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349: 132-138).