Quantifying Yolk Protein Levels in Worms

Yolk protein levels were quantified by running worm protein extracts on PAGE gels and then staining with Coomassie blue dye as described [19 (link)]. 20 worms were picked into 25 μL of M9 buffer and frozen at −80°C. Samples where then thawed, and 25 μL of 2x Laemmli sample buffer (Sigma) added. Samples were incubated at 70°C, vortexed continuously for 15 min, incubated at 95°C for 5 min and spun at 6,000 rpm for 15 min. Samples were loaded onto Criterion XT precast gels 4%–12% Bis-Tris (Bio-Rad), using XT MOPS (Bio-Rad) as a running buffer, and stained and destained following standard protocols. Gels were analyzed using ImageQuant LAS-4000 (GE Healthcare). Protein band identification was based on published data [19 (link), 21 (link)]. YP170, YP115 and YP88 were previously identified as vitellogenins by means of peptide mapping [60 (link)]. Correct identification in the present study of vitellogenins on protein gels was confirmed by the effect of vit-5,6 RNAi, which abrogated YP170, YP115 and YP88 accumulation (Figure S4A). Yolk proteins were normalized to myosin and the ratio of actin to myosin was estimated to assess the reliability of myosin as a standard for normalization [19 (link)].