Super west femto chemiluminescent substrate

After hatching, whole larvae were homogenized in PBS (0.8% NaCl, 0.02% KCl, 0.144% Na₂HPO4, 0.024% KH₂PO4, 20 mM protease inhibitor cocktail, and 20 mM PMSF, pH 7.5). The protein concentrations were measured with the Bradford method53 (link) and 150 μg of protein was separated with 6% SDS-PAGE and transferred onto PVDF membranes (Roche). The membranes were blocked with 5% nonfat dry milk overnight at 4 °C, before incubation with rabbit anti-Bmsei antibody (1:10,000) for 2 h at 37 °C, and were then incubated with goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody (1:20000, A6154; Sigma) for 1 h at 37 °C. To normalize Bmsei expression to a reference protein, the membranes were also probed with a mouse anti-α-tubulin antibody (1:20,000, AT819; Beyotime). A secondary peroxidase-conjugated anti-mouse IgG antibody produced in goats (A2554, Sigma) was used at 1:20,000. Final visualization was achieved with the Super West Femto Chemiluminescent Substrate (30945; Thermo Scientific). The blots were analyzed and quantified with the Quantity One 4.6.2 software (Bio-Rad).

Polyclonal HRG1 antibody serum was generated in rabbit using the C-terminal 17 amino acid peptide sequence (YAHRYRADFADIILSDF) of human Hrg1 as antigen (Epitomics, Inc.). Since the C-terminal 17 amino acid sequence of human Hrg1 has high homology to zebrafish Hrg1a and Hrg1b (15/17), it cross-reacts with both Hrg1a and Hrg1b.
For western blot analysis of Hrg1 protein in zebrafish, total protein concentration in membrane fractionation lysate was measured using the Pierce BCA assay kit (Thermo Scientific). Equal amount of total protein was mixed with Laemmli sample buffer and were separated on 12% SDS-PAGE and transferred to a 45 μM nitrocellulose membrane with semi-dry transfer apparatus (Bio-Rad). The affinity purified Hrg1 antibody was used at a concentration of 1:1000, goat anti-rabbit HRP-conjugated secondary was used at 1: 30,000, and blots were developed in SuperWest Femto Chemiluminescent Substrate (Thermo Scientific).