Protein Immunodetection

Protein was resolved by Laemmli SDS-PAGE on 4–15% polyacrylamide gel before electrotransfer to 0.2 µm nitrocellulose membrane in Laemmli buffer, 20% ethanol, 0.02% SDS. Protein transfer was controlled by Ponceau red staining of the membrane and positioned with Precision Plus Protein Dual Color standards (Eurogentec). The membranes were blocked in TBS, 0.1% Tween-20, 5% BSA. Immunostaining was done by incubation of the blot with specific antibodies in TBS, 0.1% Tween-20, 1% BSA, washing in TBS, 0.1% Tween-20 and detection by horseradish-peroxidase coupled reaction visualized on Chemidoc MP Imaging system (BioRad). Antibodies against ALA10 were obtained by rabbit immunization with the ALA10 C-terminus peptide RSARFHDQIYKDLVGV fused to the N-terminus of ovalbumin and affinity purification on the peptide. Antibodies were used at the following dilution: anti-ALA10 at 1/10,000, anti-GFP-HRP conjugated (Mitenyi Biotech) at 1/5,000, anti-ubiquitin (P4D1)-HRP conjugated (Cell signaling) at 1/1,000, anti-ubiquitin Lys48-specific (Millipore) at 1/1,000, anti-ubiquitin Lys63-specific (Millipore) at 1/1,000, anti SMT (Agrisera) at 1/5,000.