Fig. 2.

Autoradiogram of stoichiometric quantitation of ubiquitin conjugation components. Incubations were performed as described in Subheading 3.1 for ¹²⁵I-ubiquitin in the absence (lane 1) or presence of the indicated components. Human Uba1 and recombinant UbcH7 E2 carrier protein were present at 50 nM. Recombinant GST–E6AP Hect domain fusion protein was present at 10 nM. Shown are the autoradiographic densities for the corresponding ¹²⁵I-ubiquitin thiolesters. Quantitation of these thiolester intermediates is achieved by excising the corresponding bands and quantitating associated ¹²⁵I by gamma counting.