Goat anti mouse hrp conjugate

Worm and cell lysates were prepared in RIPA buffer (50 mM Tris-HCl pH 7.4,150 mM NaCl, 1% Triton x-100, 1% Sodium deoxycholate, 0.1% SDS,1 mM EDTA with proteinase inhibitors cocktail from Roche) unless otherwise indicated, followed by water bath sonication on maximum energy in a Diagenode Bioruptor XL 4 °C water bath at 30s/30s on and off intervals for a total of 15 min. Lysates were cleared of insoluble material by centrifugation at 21,000g at 4 °C and the supernatant was retained for western blotting. Protein concentration was determined using the Pierce BCA assay (Thermo Fisher). SDS-PAGE was conducted followed by electrophoretic transfer to nitrocellulose membrane at 100 V for 1 hour at 4 °C. Immunoblots were performed according to primary antibody manufacturers’ protocols. For immunodetection of primary antibodies, goat-anti-rabbit-HRP conjugate or goat-anti-mouse-HRP conjugate (GE Healthcare) was used at 1:5,000 in 5% BSA dissolved in TBST, and HRP was detected using West-Pico chemiluminescence substrate (Thermo Pierce). The western blot results shown are representative of at least three independent experiments.