Clarity and claritymax western blotting substrates

Samples were subjected to SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat dry milk in TBST (50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween-20) and incubated with primary antibodies as indicated. Antibodies used were: mouse anti-c-Myc 9E10 (1:1000, Thermo Fisher Scientific, Waltham, MA), mouse anti-FLAG (1:1000, Sigma Aldrich, St. Louis, MI) and mouse anti-actin (1:2000, Merck Millipore, Burlington, MA). Primary antibodies were detected using HRP coupled secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, Cambridge House, UK). Chemiluminescent signal was developed using Clarity and ClarityMax Western Blotting Substrates (BioRad Laboratories Inc., Hercules, CA) followed by detection with a FujiFilm ImageQuant LAS-4000 detector (GE Healthcare, Chicago, IL).

For protein extraction, 50–100 worms per condition were lysed directly in ice-cold 3xLaemmli buffer (6%w/v SDS, 30%glycerol, 120 mM Tris–Cl pH 6.8, 0.03% w/v bromophenol blue) and heated for 5 min at 95 °C. Samples were directly subjected to SDS-PAGE, transferred to PVDF membranes, blocked with 5% nonfat dry milk in TBST (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) and incubated with primary antibodies as indicated. Antibodies used were mouse anti-FLAG (1:2000, Sigma Aldrich, St. Louis, MI) and mouse anti-tubulin (1:20,000, Merck Millipore, Burlington, MA). Primary antibodies were detected using HRP-coupled secondary antibodies (1:10,000, Jackson ImmunoResearch Laboratories, Cambridge House, UK). The chemiluminescent signal was developed using Clarity and ClarityMax Western Blotting Substrates (BioRad Laboratories Inc., Hercules, CA) followed by detection with a FujiFilm ImageQuant LAS-4000 detector (GE Healthcare, Chicago, IL).