Anti mouse igg coupled to horseradish peroxidase

Embryos were injected at the 4 cell stage with 10 ng of Xenopus Dkk2-Flag mRNA alone or in the presence of increasing doses of Dkk2MO, and cultured to stage 13. Pools of 10 embryos were homogenized in lysis buffer (0.5% Triton X-100, 10 mM Tris–HCI at pH 7.5, 50 mM NaCl, 1 mM EDTA), containing Halt^TM Protease Inhibitor Cocktail (ThermoFisher Scientific; Waltham, MA). After two consecutive centrifugations to eliminate lipids, the lysate was concentrated on an Amicon Ultra Centrifugal Filter (Merck Millipore; Billerica, MA), 5 μl of the concentrated lysate was resolved on a 10% NuPAGE Bis-Tris gel and transferred onto a PVDF membrane using the iBlot system (Invitrogen). Blots were subsequently incubated overnight with one of the following primary antibodies: monoclonal anti-Flag M2 antibody (Sigma Aldrich, F3165; 1:1000 dilution) and anti α-tubulin antibody (Sigma Aldrich, T9026; 1:500 dilution). The blots were then washed and incubated with anti-mouse IgG coupled to horseradish peroxidase (Santa Cruz Biotechnology; 1:10,000 dilution). Peroxidase activity was detected with the Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and imaged on a ChemiDoc MP Biorad gel documentation system (Hercules, CA). Membranes were stripped using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) according to the manufacturer recommendations.