Anti tp53 antibody

In total, 21 embryos were divided into 3 groups (each n=7) for 48 hpf wild-type and banp^rw337 mutants, respectively. Heads were dissected in ice-cold E3 medium. Seven heads removed from each group were homogenized together in sample buffer (125 mM NaCl, 50 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 1% Triton X-100, 1 X Protease inhibitor) and used as one sample. Appropriate volumes of samples were diluted to provide equal protein amounts and used for SDS-PAGE (BIO-RAD, Mini-PROTEAN TGX Gels). Western blot analysis was performed on 3 samples for wild-type and banp^rw337 mutants, respectively, according to standard protocols using anti-tp53 antibody (GeneTex, GTX128135) at a 1:1000 dilution. β-actin protein level was evaluated by western blot analysis of rehybridized membranes with anti-β-actin antibody (Sigma, A5441) at a 1:5,000 dilution, and used to normalize tp53 protein level for each sample. For secondary antibodies, horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Amersham, NA931 and NA934) were used. Enzyme activity was detected using Immunostar LD (Wako) and luminescence was imaged and quantified using iBright 1,500 (Thermo Fisher Scientific).