Protein was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a Bicinchoninic Acid Assay kit (Beyotime Institute of Biotechnology). A total of 40 µg of protein was used for 10% SDS-PAGE and transferred to PVDF membranes. Subsequently, the membranes were blocked with 5% skim milk at 4°C overnight. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-ANP (1:1,000; cat. no. A14755; ABclonal Biotech Co., Ltd.), anti-NFAT2 (1:1,000; cat. no. A1539; ABclonal Biotech Co., Ltd.), anti-α-actinin (1:1,000; cat. no. sc-17829; Santa Cruz Biotechnology, Inc.), anti-β-MHC (1:1,000; cat. no. A7564; ABclonal Biotech Co., Ltd.), anti-TRPC3 (1:1,000; cat. no. A7742; ABclonal Biotech Co., Ltd.), anti-TRPC6 (1:1,000; cat. no. bs-2393R; BIOSS) and anti-GAPDH (1:2,000; cat. no. AC033; ABclonal Biotech Co., Ltd.). Subsequently, the membranes were incubated with a horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin (Ig)G secondary antibody (1:5,000; cat. no. bs-0295G-HRP; BIOSS) or HRP-conjugated goat anti-mouse IgG secondary antibody (1:5,000; cat. no. AS003; ABclonal Biotech Co., Ltd.) for 2 h at room temperature. The membranes were detected with Immobilon Western HRP substrate (EMD Millipore). The bands were semi-quantified by ImageJ 2× software (Rawak Software, Inc.).
Anti β mhc
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-β-MHC is a laboratory reagent used in research applications. It is an antibody that specifically binds to the beta-myosin heavy chain (β-MHC) protein. The core function of Anti-β-MHC is to facilitate the detection and study of β-MHC in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Anti gapdh, by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid assay kit, by Beyotime (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
Eclipse e100 microscope, by Nikon (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti α mhc, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
Anti bnp, by Santa Cruz Biotechnology (1 mentions)
Skim milk, by Thermo Fisher Scientific (1 mentions)
Amersham image 600, by GE Healthcare (1 mentions)
Anti anp, by Santa Cruz Biotechnology (1 mentions)
Image pro plus 6, by GE Healthcare (1 mentions)
4 protocols using anti β mhc
1
Cardiac Protein Expression Analysis
2
Quantifying Cardiac Protein Levels
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The myocardial expression levels of α- and β-myosin heavy chain (MHC), Bax, as well as Bcl-2 were detected using immunohistochemistry techniques. Sections (10 μm) from paraffin-embedded tissues were stained using the simplified histostain SP-kit (Zymed Laboratories, USA) according to the manufacturer's instructions. After deparaffinization and rehydration and the inhibition of endogenous peroxidase, the sections were exposed to primary antibodies at 4°C overnight. After incubation with a secondary antibody at room temperature, the sections were incubated in 3,3 N-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. The following primary antibodies were used at a dilution of 1:150: anti-α-MHC, anti-β-MHC, anti-Bax and anti-Bcl-2 (Santa Cruz Biotechnology, CA, USA). The secondary antibody was goat-anti-mouse immunoglobulin conjugated with horseradish peroxidase (dilution of 1:200). Digital images of the stained sections were acquired using a Nikon ECLIPSE E100 microscope with a digital camera system. Unanimity regarding positive immunohistochemical staining in each preparation was reached by two blinded investigators using Image-Pro Plus 5.1 software (Media Cybernetics, Silver Spring, MD).
3
Western Blot Analysis of MURC, β-MHC, and BNP
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We used Western blotting to detect protein levels as previously described.12 The antibodies used for the Western blot analysis were anti‐MURC (1:1000 dilution; Sigma‐Aldrich, St. Louis, MO, USA), anti‐β‐MHC (β‐myosin heavy chain) and anti‐BNP (B‐type natriuretic peptide) (1:200 dilution; both from Santa Cruz Biotechnology, Dallas, TX, USA).
4
Cardiomyocyte Protein Expression Analysis
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Total protein was extracted from cultured neonatal cardiomyocytes and protein concentration was measured using a BCA protein assay kit (Thermo Scientific, USA). Protein samples were separated by 8% or 12% acrylamide denaturing gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. Following transfer, membranes were washed with tris-buffered saline and polysorbate 20 (TBST), and then blocked in 5% skim milk (Gibco, USA) in TBST for 2 h at room temperature, followed by incubation with anti-ANP (1:1000 dilution, Santa Cruz Biotechnology), anti-β-MHC (1:1000 dilution, Santa Cruz Biotechnology), anti-phospho-Smad3 antibody (p-Smad3, 1:1000 dilution, CST), anti-Smad3 antibody (1:1000 dilution, CST) and anti-GADPH (1:10,000 dilution, BOSTER) antibodies on a shaker overnight at 4 °C. The following day, membranes were washed in TBST and then incubated with secondary antibodies (1:8000 dilution, BOSTER) for 2 h at room temperature. Antibody-antigen complexes in all membranes were detected using the Imaging System (GE, Amersham Image 600) and protein bands were quantified by Image Pro Plus 6.0.
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