Proteins were extracted from yeast, cultured mammalian cells, or plant roots, as detailed in the Materials and Methods section. The shaded box highlights the common stages of the purification, which included a heating step and removal of precipitated proteins by centrifugation. The composition of the purified unanchored polyubiquitin was analysed by western blot analyses following elution with gel loading buffer. Ubiquitin and K48-polyubiquitin linkage specific antibodies were used for western blot analysis.
