Western Blot Protein Detection Protocol

Proteins were transferred to 0.45 μm Immobilon-P PVDF membrane (Millipore, Burlington, MA) in transfer buffer (25 mM Tris, 200 mM Glycine, 20% methanol) for 60 min (75 min for Zld) at 500mA at 4°C. The membranes were blocked with blotto (2.5% non-fat dry milk, 0.5% BSA, 0.5% NP-40, in TBST) for 30 min at room temperature and then incubated with anti-GFP (1:2000, #ab290) (Abcam, Cambridge, United Kingdom) anti-Zld (1:750) (Harrison et al., 2010 (link)), or anti-tubulin (DM1A, 1:5000) (Sigma, St. Louis, MO), overnight at 4°C. The secondary incubation was performed with goat anti-rabbit IgG-HRP conjugate (1:3000) (Bio-Rad, Hercules, CA) or anti-mouse IgG-HRP conjugate (1:3000) (Bio-Rad) for 1 hr at room temperature. Blots were treated with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) and visualized using the Azure Biosystems c600 or Kodak/Carestream BioMax Film (VWR, Radnor, PA).