Figure 5. Monomeric ubiquilin-1 binds PS1 and PS2 in vitro.

Purified proteins GST–ubiquilin-1 and ubiquilin-1–His₆ were mixed together in vitro along with [³⁵S]methionine-labelled PS1 or PS2. The protein mixture was sequentially passed over glutathione–agarose and Ni-NTA columns. Unbound protein was collected from each column prior to washing with the appropriate column wash buffer. The eluted (bound) protein mixture from the glutathione–agarose column was subsequently added to the Ni-NTA column. The bound protein mixture was eluted from each column using 10 mM GSH or 250 mM imidazole buffer. Protein fractions were separated by SDS/PAGE. Autoradiography revealed that PS1 binds to the glutathione–agarose column, but not the subsequent Ni-NTA column. Immunoblotting the [³⁵S]methionine-labelled PS1 blot for ubiquilin revealed that GST–ubiquilin-1 and ubiquilin-1–His₆ both remain after passing through the two columns. Ubqln, ubiquilin.