Immunofluorescence and Western Blot Protocols

Anti-Marshalin21 (link) was used at 2 μg/ml for immunofluorescence (IF) and 0.6 μg/ml for Western blot. Commercial antibody dilutions were as follows: anti-V5 (Invitrogen), 1:1,000 dilution for IF and 1:5,000 for Western blot; anti-α-tubulin (Zymed, San Francisco, CA), 1:800 for IF; anti-GFP (Clontech, Mountain View, CA), 1:2,000 for Western blot; anti-GST (Sigma), 1:10,000 for Western blot; goat anti-mouse IgG-Alexa Fluor 546, goat anti-rabbit IgG-Alexa Fluor 488 or 546 (Molecular Probes, Eugene, OR), 1:500 for IF; goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (Jackson ImmunoResearch, West Grove, PA), 1:10,000 for Western blot; Texas Red-X phalloidin (Molecular Probes, Eugene, OR), 1:2,000 to stain F-actin. Donkey anti-mouse IgG-Alexa Fluor 647F(ab’)₂ Fragment (Jackson ImmunoResearch, West Grove, PA), 1:1,000 for IF.