Horseradish peroxidase hrp conjugated goat anti mouse antibody

ZFN-injected and uninjected embryos were sampled at 3, 6, 9 and 24 hours post-injection to determine the temporal profile of ZFN protein expression. At each time point, one pool of 10 embryos was sampled. Embryos were homogenized in 1× sample treatment buffer with β-mercaptoethanol and boiled for 5 minutes to denature the protein. Samples were separated on an 8% polyacrylamide gel using the discontinuous buffer system (Laemmli, 1970 (link)). The proteins were transferred onto a nitrocellulose membrane with a semi-dry transfer unit (BioRad, Waltham, MA, USA), and the membrane was blocked with 5% milk in TBST (20 mM Tris pH 7.5, 300 mM NaCl and 0.1% (v/v) Tween 20) overnight. ZFN protein was detected using a mouse monoclonal anti-Flag antibody (Sigma, St. Louis, MO, USA) at 1:5000 dilution. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at 1:5000 dilution. The membranes were incubated in the primary antibody for 60 min at room temperature, washed with TBST twice (10 min each), incubated with secondary antibody for 60 min, and finally washed with TBST (15 min). Detection was done using the ECL Prime Western blotting reagent (GE Healthcare, Pittsburgh, PA, USA).