Anti myhc

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-MyHC is a laboratory product developed by Santa Cruz Biotechnology for use in research applications. It is an antibody that specifically binds to and detects myosin heavy chain (MyHC), a key structural protein found in muscle tissue. This product can be used to identify and study muscle cells and their development in various research contexts.

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Lab products found in correlation

Fluorescence microscope, by Olympus (3 mentions) Affinipure goat anti mouse igg, by ZenBio (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Goat serum, by Beyotime (2 mentions) Primescript rt kit, by Takara Bio (1 mentions) Anti tubulin, by Merck Group (1 mentions) Anti hsp90, by Cell Signaling Technology (1 mentions) Anti cyclin d1, by Santa Cruz Biotechnology (1 mentions) Anti sp1, by Santa Cruz Biotechnology (1 mentions) Anti pax7, by Santa Cruz Biotechnology (1 mentions) Anti mef 2c, by Santa Cruz Biotechnology (1 mentions) Anti myog, by Santa Cruz Biotechnology (1 mentions) Anti p21, by Santa Cruz Biotechnology (1 mentions) Abi 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions) Anti gapdh, by Merck Group (1 mentions) Anti caspase 9, by Abcam (1 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Dapi 4 6 diamidino 2 phenylindole, by Solarbio (1 mentions)

7 protocols using anti myhc

Western Blot and qPCR Analysis

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Radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.0 mM EDTA, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton X-100) was used to prepare cell or frozen tissue protein lysates. The protein concentration was determined by using BCA protein assay kit (Thermo) following the instructions. Protein lysates were resolved on 10% SDS‒PAGE gels using standard procedures. Anti-Sp1 (106 kDa; Santa Cruz), anti-Pax7 (44‒50 kDa; Santa Cruz), anti-Cyclin D1 (37 kDa; Santa Cruz), anti-MEF-2C (40‒65 kDa; Santa Cruz), anti-MyoG (34 kDa; Santa Cruz), anti-MyHC (200 kDa; Santa Cruz), anti-p21 (21 kDa; Santa Cruz), anti-Tubulin (55 kDa; Sigma Aldrich), anti-GAPDH (36 kDa; Sigma Aldrich), and anti-HSP90 (90 kDa; Cell Signaling Technology) were used for western blot analysis.
Trizol reagent (Thermo) was used to extract total RNA from cells or frozen tissues according to the standard procedure. First strand cDNA was synthesized by using the PrimeScript RT kit (TaKaRa). The miRNAs were reverse transcribed by stem-loop RT system, which was performed as described previously (Liu et al., 2014 (link)). qPCR was performed on an ABI7900 Real Time PCR System (Applied Biosystems). The detailed information of PCR primers is in Supplementary Table S1.

Wang Y.C., Yao X., Ma M., Zhang H., Wang H., Zhao L., Liu S., Sun C., Li P., Wu Y., Li X., Jiang J., Li Y., Li Y, & Ying H. (2021). miR-130b inhibits proliferation and promotes differentiation in myocytes via targeting Sp1. Journal of Molecular Cell Biology, 13(6), 422-432.

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Quantifying Myotube Formation in Chicken Satellite Cells

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Chicken skeletal muscle satellite cells were seeded in 24-well plates, cultured in DM and transfected as described in section “Vector construction and RNA oligonucleotides”. After 72 h, cells were fixed in 4% formaldehyde for 30 min then washed three times with PBS for 5 min. Subsequently, the cells were permeabilized by adding 0.1% Triton X-100 for 20 min and blocked with 5% goat serum (Beyotime) for 30 min. After incubation with anti-MyHC (Santa Cruz; 1:250) at 4°C for 12 h, the Rhodamine (TRITC) AffiniPure Goat Anti-Mouse IgG (ZenBio; 1:1000) was added and the cells were incubated at 37°C for 1 h. The cell nuclei were stained with DAPI (Beyotime; 1:50) for 5 min. Three images were obtained at random using a fluorescence microscope (Olympus, Japan). The area of myotubes was measured by Image-Pro Plus software.

Yin H., Shen X., Zhao J., Cao X., He H., Han S., Chen Y., Cui C., Wei Y., Wang Y., Li D, & Zhu Q. (2020). Circular RNA CircFAM188B Encodes a Protein That Regulates Proliferation and Differentiation of Chicken Skeletal Muscle Satellite Cells. Frontiers in Cell and Developmental Biology, 8, 522588.

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Quantifying Chicken Myoblast Differentiation

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Chicken primary myoblasts were seeded in 48-well plates, and the cells were induced to differentiation for 72 h after transfected by treatments. The cells were fixed in 4% formaldehyde for 30 min and permeabilized by 0.1% Triton X-100 for 20 min and then blocked by 5% goat serum (Beyotime) at room temperature for 30 min. Thereafter, the cells were incubated with primary antibody anti-MyHC (Santa Cruz; 1:250) at 4°C for 12 h and then incubated with the Rhodamine (TRITC) AffiniPure Goat Anti-Mouse IgG (ZenBio; 1:1,000) at 37°C for 1 h. Finally, cell nucleus was stained with DAPI (Beyotime; 1:50) for 5 min. Three images were randomly captured by a fluorescence microscope (Olympus, Japan). The MyHC labeled myotube area and DAPI-labeled nuclear area (Internal control) were measured by Image-Pro Plus software, and the ratio of myotube area to nuclear area is taken as the relative myotube area.

Shen X., Wei Y., Liu W., You G., Tang S., Su Z., Du M., He J., Zhao J., Tian Y., Zhang Y., Ma M., Zhu Q, & Yin H. (2021). A Novel Circular RNA circITSN2 Targets the miR-218-5p/LMO7 Axis to Promote Chicken Embryonic Myoblast Proliferation and Differentiation. Frontiers in Cell and Developmental Biology, 9, 748844.

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Immunofluorescence and Western Blot Analysis

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Cells were fixed using 4% paraformaldehyde (Solarbio) according to the manufacturer’s instructions. The fixed cells were infiltrated with 0.5% Triton X-100 (Beyotime, Shanghai, China) for 20 min after washing with PBS (Hyclone) and blocked with goat serum (Solarbio) for 30 min. Next, the primary antibody was added and incubated overnight at 4 °C. The cells were washed with PBST (0.05% Tween 20; Chron Chemical, Chengdu, Sichuan, China) + PBS (Hyclone, South Logan, UT, USA) and a diluted secondary antibody was added and incubated at 37 °C for 1 h. Next, the nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole; Solarbio) for 5 min in the dark. Images were captured by a fluorescence microscope (Olympus, Melville, NY, USA).
Detailed experimental methods for the Western blot analysis are described in depth by Shen et al. (2019) [45 (link)]. The antibodies used for the experiments were as follows: Anti-MyHC (Santa Cruz Biotechnology, CA, USA; 1:200 dilution), anti-MyoD (Santa Cruz Biotechnology; 1:500 dilution), anti-caspase-3 (Abcam, London, UK; 1:1,000 dilution),anti-caspase-9 (Abcam, London, UK; 1:1000 dilution), anti-TGF-β2 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution), anti-β-actin (Santa Cruz Biotechnology; 1:1000 dilution), anti-SMAD2/3 and anti-p-SMAD2/3 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution). β-actin was used as the loading control.

Yin H., He H., Shen X., Tang S., Zhao J., Cao X., Han S., Cui C., Chen Y., Wei Y., Wang Y., Li D, & Zhu Q. (2020). MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-β2 and Regulating the TGF-β2/SMAD Signaling Pathway. International Journal of Molecular Sciences, 21(9), 3274.

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Immunostaining of Differentiated Myotubes

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For immunostaining, differentiated C2C12 myotubes or primary myotubes were fixed in 4% paraformaldehyde and incubated with 0.3% Triton X-100 to enhance permeability. Fixed samples were blocked with 3% bovine serum albumin in PBS and treated with anti-MyHC (Santa Cruz Biotechnology), followed by washing in PBS and incubation with Alexa Fluor 488 (Invitrogen) secondary antibodies. The fusion index was determined by dividing the number of nuclei in MyHC-positive myotubes with ≥2 nuclei by the total number of nuclei analyzed (38 (link)).

Lee B., Shin Y.J., Lee S.M., Son Y.H., Yang Y.R, & Lee K.P. (2020). miR-3074-3p promotes myoblast differentiation by targeting Cav1. BMB Reports, 53(5), 278-283.

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Quantitative Protein Analysis in Muscle

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MuSC proteins were obtained by the total protein extraction kit (Beyotime, Shanghai, China). Proteins were denatured (100 °C, 5 min) by adding protein loading buffer (5× SDS-PAGE, Beyotime). ImageJ software (version 1.53; https://imagej.nih.gov/ij/; (accessed on 24 September 2021)) was used to perform the semi-quantitative study of proteins. The WB used the following antibodies: anti-β-tubulin (1:1000, 250007, ZENBIO, Chengdu, China), anti-MyHC (1:1000, sc-378137, Santa Cruz, Dallas, TX, USA), anti-MEF2C (1:500, 10056-1-AP, Proteintech, Wuhan, China), anti-YTHDC1 (1:1000, ab220159, Abcam, Cambridge, MA, USA), anti-METTL3 (1:1000, A8370, ABclonal, Wuhan, China), MyoD (1:1000, 18943-1-AP, Proteintech, Proteintech, Wuhan, China), anti-Mouse IgG (1:5000, 511103, ZENBIO, Chengdu, China), and anti- Rabbit IgG (AS014, 1:5000, ABclonal, Wuhan, China).

Zhao S., Cao J., Sun Y., Zhou H., Zhu Q., Dai D., Zhan S., Guo J., Zhong T., Wang L., Li L, & Zhang H. (2023). METTL3 Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating MEF2C mRNA Stability in a m6A-Dependent Manner. International Journal of Molecular Sciences, 24(18), 14115.

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GPRC6A Regulation by siRNA Knockdown

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Cells were lysed for 30 min on ice in radioimmunoprecipitationassay (RIPA) lysis buffer (Solarbio, Beijing, China) containing 0.1mM PMSF and a protease inhibitor (Roche).The samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed using primary antibodies, including anti-phospho-Akt (Cell Signaling Technology, CST, Boston, MA, USA), anti-Akt (CST), anti-phospho-P38 (CST), anti-P38 (CST), anti-phospho-ERK1/2 (CST), anti-ERK1/2 (CST), anti-GPRC6A (SantaCruz), anti-MyHC (Santa Cruz), and anti-GAPDH (CST). The lysates were subjected to standard Western Blotting analysis. The images were obtained usingTanon-5200 (Tanon, Shanghai, China). The relative densities of the specific bands were quantified using the image detection software Image Lab 5.0 (Bio-Rad Laboratories Inc., California, USA).
Short interfering RNA (siRNA) and transfection siRNA transfection was used to downregulate GPRC6A expression. All the siRNAs were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). The GPRC6A-targeted siRNA (si GPRC6A) and control siRNA (si Ctrl) were dissolved separately in Optimem I (Invitrogen). After 10 min of equilibration at room temperature, each RNA solution was combined with an equal volume of Lipofectamine™ 2000 solution, mixed gently, and allowed to form siRNA liposomes for 20 min.

Liu S., Gao F., Wen L., Ouyang M., Wang Y., Wang Q., Luo L, & Jian Z. (2017). Osteocalcin Induces Proliferation via Positive Activation of the PI3K/Akt, P38 MAPK Pathways and Promotes Differentiation Through Activation of the GPRC6A-ERK1/2 Pathway in C2C12 Myoblast Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 43(3).

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