Western Blot Analysis of Ulp1-GFP Derivatives

Ulp1-GFP and various ulp1Δ-GFP derivatives were detected using western blot. Proteins from cells lysates were separated by SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were incubated in blocking buffer (TBS containing 0.1% Tween-20 and 5% milk powder) for at least 1 h at room temperature. Blocking buffer was then removed and replaced with fresh blocking buffer supplemented with rabbit polyclonal antibodies directed against GFP, GSP1, or SUMO (Makhnevych et al., 2007 (link)) then incubated overnight at 4°C. Membranes were then washed three times using 0.1% Tween-20 in TBS, followed by incubation in blocking buffer supplemented with goat anti-rabbit HRP conjugated antibody (BioRad) at a 1:10,000 dilution for at least 1 h at room temperature. Membranes were then washed three times using 0.1% Tween-20 in TBS. Bound anti-rabbit HRP conjugated antibody was detected by chemiluminescence (Amersham) using an ImageQuant LAS 4000 (GE) imaging system.