Deyolking and Lysis of Zebrafish Embryos

Heat shocked and dechorionated embryos were collected at 3dpf. To deyolk embryos, a borosilicate injection needle was used to mechanically disrupt yolks. Embryos were next washed in deyolking buffer without calcium (Link et al., 2006 (link)), spun at 300rcf and washed in wash buffer (110mM NaCl, 3.5mM KCl, 2.7mM CaCl₂, 10mM Tris/Cl) containing Complete Mini protease inhibitor mixture (Roche Diagnostics). Deyolked embryos were lysed with modified LeMeer’s Lysis Buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1% IGEPAL, 0.1% sodium deoxycholate) supplemented with Complete Mini protease inhibitor cocktail (Lemeer et al., 2007 (link)) before being centrifuged at low speed and sonicated by a Sonic Dismembranator Model 300 (Fisher Scientific). Using manufacturer’s protocol, protein samples were gel electrophoresed using 4–12% Bis-Tris gel and transferred onto PVDF membrane (Invitrogen NuPage system). Blots were incubated 1:5000 anti-myc (abcam ab9106), followed by 1:5000 horse anti-mouse HRP secondary (Cell Signaling Technology 7076). Blots were imaged via the Super Signal West Femto visualization system (Life Sciences) on an ImageQuant LAS4000 machine (GE Life Sciences). Following imaging of myc antibody labeling, blots were stripped for 15 minutes in Restore Western Blot Stripping Buffer (Thermo Scientific 21059) and reprobed with 1:5000 anti-actin (calbiochem cp01).