HeLa Cell Protein Analysis Protocol

HeLa Kyoto and derivatives were lysed into a buffer containing 20mM Tris-HCl, pH 8.0, 100mM KCl, 1% Triton X-100 (Splinter et al., 2012 (link)), benzonase (25 U/mL) and a protease-inhibitors cocktail (Protease-inhibitor mix HP Plus, Serva). Lysates were diluted at 0.5 mg/ml into Laemmli buffer, boiled at 95°C and resolved on a 6% SDS-PAGE before transfer onto a nitrocellulose membrane. After blocking with 5% milk in PBS-Tween (0.1%), membranes were probed with antibodies diluted as follows: anti-alpha-tubulin DM1α, 1:5000 (Sigma-Aldrich); rabbit polyclonal anti-GFP, 1: 5000 (Abcam); mouse anti-p150^Glued, 1:1000 (BD Transduction Laboratories); Sheep anti-mouse HRP and Donkey anti-rabbit HRP, 1:5000 (Amersham). All membranes were incubated with ECL reagent and developed onto Hyperfilm (Amersham) or imaged with the ChemiDoc™ MP Imaging System (BIO-RAD). The levels of images from scanned Hyperfilm and ChemiDoc tiff-converted files were adjusted using FIJI and converted to 8-bit. Western blots against the dynein HC were performed as above with the following modifications; lysates were diluted to 0.75 - 1mg/ml in Laemmli buffer, blocking buffer was 5% BSA in PBS-Tween (0.1%), membranes were probed with Rat dynein HC antibody (Santa Cruz Biotechnology) at 1:250 dilution.