Monitoring ATX3-Q55 Protein Production by Western Blot

The production of ATX3-Q55 was monitored by WB analysis. Crude extracts were obtained from aliquots of OD₆₀₀ ~ 4 of cells collected at different growth times according to the procedure previously described [28 (link)] and denatured in the Laemmli buffer as previously described. A preliminary SDS-PAGE analysis was carried out to determine the amount of crude extracts used for WB analysis. Comparable amount of crude extracts (~ OD₆₀₀: 0.20) was loaded on 12% SDS-PAGE and then blotted on Odyssey nitrocellulose membrane (LI-COR Biosciences, Lincoln, NE, USA) using the Mini Protean electroblotting system (Bio-Rad, Hercules, CA, USA) and Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3) (constant current 400 mA for 2 h at 4 °C). Rabbit polyclonal primary anti-AT3 Z46 (1:5000) and anti-PNPase (1:150,000) antibodies were diluted in 5% milk and incubated overnight; anti-rabbit fluorescent IRDye^® 800CW (LI-COR Biosciences, Lincoln, NE, USA) was used as a secondary antibody for both primary antibodies. Signal quantification was carried out using Image Studio software (LI-COR Biosciences, Lincoln, NE, USA). The experiment was performed in triplicate.