Hrp conjugated goat anti rabbit igg

Crude protein was extracted from 1 g of gill tissue with cell lysis buffer (Beyotime, Shanghai, China). After concentration determination with a BCA protein assay kit (Promega, Madison, WI), protein expression patterns were measured by Western blotting. Quantification was accomplished with relative antibodies as follows: (a) 20 μg of crude protein was separated using SDS-PAGE and then transferred to a PVDF membrane (Merck, USA). (b) The membranes were blocked in TBST buffer (Solarbio, Beijing, China) with 5% skim milk (Solarbio, Beijing, China) for one hour at room temperature. (c) The blocked membranes were incubated with diluted primary antibody (1:1000) for 12 h at 4 °C. (d) The membranes were conjugated with anti-rabbit immunoglobulin horseradish peroxidase-conjugated secondary antibody at a dilution rate of 1:5000 for one hour. (d) Proteins were visualized with ECL detection reagent (Beyotime, Shanghai, China). The primary antibodies for ANXA1, NDRG1, BAX, MMP9 (Art. No: WL0040; WL03071; WL02635; WL03096) and HRP-conjugated Goat Anti-Rabbit IgG (Art. No: WLA023) were from WanleiBio (Shenyang, China). Primary antibodies of β-actin was from Boster (Art. No: BM5422, Wuhan, China) and corresponding HRP-labeled secondary antibodies were from ZSGB-BIO (Art. No: ZB2305, Beijing, China).