Alexafluor680 conjugated anti mouse secondary antibody

Protein levels of KIF1A were assessed by lysing control and siRNA expressing dissociated cortical cultures in 50 μL of phosphate buffered saline with cOmplete, ethylenediaminetetraacetic acid (EDTA)‐free protease inhibitor cocktail tablets (Sigma‐Aldrich). Protein samples were vortexed and then heated in Laemmli buffer to 100°C for 10 min. A total of 15 µl of each sample were separated on a 12% SDS‐PAGE. After transferring the gel to PVDF membrane, the membrane was probed with a polyclonal anti‐KIF1A antibody (Abcam; 1:1000) and a monoclonal anti‐beta tubulin antibody (Sigma; 1:2000). After three washes with PBST for 10 minutes each, bands were detected with AlexaFluor680‐conjugated anti‐mouse secondary antibody (Molecular Probes, Eugene, Oregon). Membranes were then scanned and quantified using the Odyssey imaging system (LI‐COR Biosciences, Lincoln, North Carolina).

SDS-PAGE and Western blotting analysis were performed as described (1 (link)). The following antibody concentrations were used: mouse monoclonal anti-FLAG (Sigma, M2), 1:3,000; mouse monoclonal anti-GFP (Roche Applied Science), 0.1 μg/ml; and Alexa Fluor680-conjugated anti-mouse secondary antibody (Molecular Probes), 1:5,000. Blots were scanned and quantified using the Odyssey imaging system (LI-COR Biosciences).