The testicular tissues of at least five 15-week-old mice per genotype were removed and homogenized in a RIPA lysis buffer containing a 1 μL/mL protease inhibitor cocktail (Sigma) to obtain cell lysates. After centrifugation, the supernatants were examined for protein concentration, subjected to 12% SDS-PAGE and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA, USA). The membranes were incubated with anti-Znf230 primary antibody (Catalog number: ab4542, Abcam, Cambridge, MA, USA) at 4 °C overnight, washed with 1 X PBS containing 0.1% Triton X-100, and incubated with horseradish peroxidase-linked secondary antibodies (1:10,000, Boster, Wuhan, China) at room temperature for 1 h. The respective bands were visualized using an immunostaining kit (Millipore). The anti-Znf230 primary antibody recognizes amino acids 218–230 in the C-terminus of the Znf230 protein. The quality of the antibody was validated by the manufacturer and our previous report (Song et al., 2008 (link)). The western blot analysis was repeated again.
Horseradish peroxidase linked secondary antibodies
Manufactured by Boster Bio
Sourced in China
Horseradish peroxidase-linked secondary antibodies are a type of laboratory reagent used in immunoassays and other immunochemical techniques. They function as a detection system, binding to primary antibodies and enabling the visualization or quantification of target analytes through an enzymatic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p transfer membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
Anti hmgcr, by Abcam (1 mentions)
Anti ldlr, by Abcam (1 mentions)
Anti srebp2, by Abcam (1 mentions)
Anti hbsag, by Abcam (1 mentions)
Anti lamin b, by Boster Bio (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
2 protocols using horseradish peroxidase linked secondary antibodies
1
Western Blot Analysis of Znf230 Protein
2
Western Blot Analysis of Cellular Proteins
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A NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Rockford, IL) was used to extract nuclear and cytoplasmic proteins according to the manufacturer’s protocol. Each 20 μg protein sample was separated on an 8/10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Darmstadt, Germany) using the Trans-Blot Turbo™ blotting system (Bio-Rad). After transferring, membranes were blocked with 5% BSA at room temperature for 2–3 h. The membranes were then incubated overnight at 4°C with anti-SREBP-2 (1:600; Abcam, Cambridge, UK), anti-HMGCR (1:5,000; Abcam), anti-LDLR (1:500; Abcam), anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) (1:600; Proteintech, Wuhan, China), anti- CYP-7α (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HBsAg (1:1,000; Abcam), anti-HBV xAg (1:200; Santa Cruz Biotechnology), anti-Hep B cAg (1:200; Santa Cruz Biotechnology), anti-GAPDH (1:10,000; Abcam), or anti-lamin B (1:300; Boster, Wuhan, China) primary antibodies, respectively. After washing with TBST, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (1:5,000; Boster). An enhanced chemiluminescence Western blotting substrate kit (BioVision, SanFrancisco, CA) was used to detect specific proteins.
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