Western Blot Analysis of Transgene Expression

M-PER mammalian protein extraction reagent (Thermo Scientific, USA) was used to
lyse the human cell lines 24 hr post-transfection, zebrafish embryos at 24 hour
post-fertilization (hpf) and mouse liver 3 day post-injection (dpi). Each lysate
was separated by 12% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Pall
Corporation; NY, USA). Subsequently, the membrane was probed with the indicated
primary antibody (anti-EGFP [1∶1000, Santa Cruz Biotechnology,
catalog # sc-9996] and anti-DsRed [1∶1000, Clontech, catalog #
632393]), washed with TBST (0.2 M Tris, 1.37 M NaCl,0.1% Tween-20,
pH7.6), probed with HRP-conjugated goat anti-mouse antibody (1∶4000, Santa
Cruz Biotechnology, catalog # sc-2005). The bound antibody was detected by
enhanced chemiluminescence (AniGen, Korea) and then exposed to X-ray film (AGFA,
Belgium).
Because anti-DsRed antibody has been successfully used to decorate mCherry
protein [12] (link),
anti-DsRed antibody was used to visualize the mCherry protein.