Anti-myogenin and anti-PIP5K1α were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), anti-GAPDH was purchased from Ambion (Austin, TX, USA), anti-phospho-AKT (T308/S473) and anti-total-AKT were purchased from Cell Signaling (Danvers, MA, USA), and anti-MHC was purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). For immunostaining, C2C12 cells were plated on six-well plates with glass coverslips. After cotransfection with siRNA for 24-hour and then 48-hour treatment of differential medium, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized by 0.2% Triton X-100 for 15 min. Cells were rinsed in PBS, blocked in 5% BSA for 1 hour, and then incubated with Anti-myogenin and anti-MHC antibody (1:500) overnight. Cells were washed three times in PBS and incubated with fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 1 hour. Then 100 ng/ml of DAPI was added for another 10 min to stain the nuclei. After three washes in PBS, the coverslips were mounted and cells visualized using an Olympus IX70 fluorescence microscope. Western blot analysis was performed according to procedures described previously [21 ].
Anti mhc
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
The Anti-MHC product from Developmental Studies Hybridoma Bank is a laboratory reagent used for the detection and analysis of major histocompatibility complex (MHC) proteins. It provides a reliable and specific tool for researchers studying the role of MHC in immune system function and cellular processes. The product description is limited to its core function as a laboratory tool, without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Anti myogenin, by Santa Cruz Biotechnology (2 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Ix70 fluorescence microscope, by Olympus (1 mentions)
Fluorescein conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Tra 1 60, by Merck Group (1 mentions)
Anti ctnt, by Merck Group (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Anti pan cadherin, by Merck Group (1 mentions)
Nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti kir2, by Santa Cruz Biotechnology (1 mentions)
Anti cdo, by R&D Systems (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti myod, by Santa Cruz Biotechnology (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Roche (1 mentions)
Anti arp5, by Proteintech (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti mhc
1
Immunostaining and Western Blot Analysis
2
Quantifying FSHD Myotube Differentiation
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96 h after differentiation, mature myotubes derived from FSHD primary myoblast were fixed with 4% paraformaldehyde for 10 min at 4°C temperature and washed with PBS. Cells were incubated with blocking buffer containing 5% Goat serum and 0.1% Triton X-100 for 1 h. Then, cells were incubated with primary antibody anti-MHC (Developmental Studies Hybridoma Bank, anti-myosin heavy chain, MF-20) for 45 min at RT, washed 3 times with PBS and incubated with secondary antibody (Thermo Fisher Scientific, goat anti-mouse Alexa fluor 488, #A32723) and DAPI for 45 min. Fluorescent images were taken by a Carl Zeiss AxioImage M2 fluorescent microscope (Zeiss, Germany). The differentiation index was calculated as the frequency (percentage) of nuclei inside Mhc-positive cells in comparison with the total number of nuclei. The fusion index was calculated as the frequency (percentage) of nuclei inside myotubes (Mhc-positive syncytia containing at least three nuclei) in comparison with the total number of nuclei. The nuclei distribution was calculated as the frequency (percentage) of Mhc-positive cells containing the indicated number of nuclei. Three independent differentiation experiments were performed and 5 fields per well were analyzed for each sample/experiment.
3
Pluripotency Assessment of Expanded hESCs
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To test the pluripotency of expanded hESCs, FACS was performed with the expression of the stem cell markers Tra-1-60 (Millipore, Darmstadt, Germany) and mAb84 [34 (link)] by flow cytometry as described in our previous studies [33 (link), 35 (link)].
CM differentiation efficiency was quantified by the expression of myosin heavy chain (MHC) and cTnT I. Briefly, cells harvested from day 20 were fixed and permeabilised using Fix and Perm Cell permeabilisation reagents (Life Technologies). The cells were subsequently incubated with anti-MHC (dilution 5:200; Developmental Studies Hybridoma Bank, Iowa city, IA, USA) and anti-cTnT (dilution 1:200; Millipore) for 20 minutes. After washing with 1% bovine serum albumin/PBS, the cells were incubated in the dark with 1:500 dilutions of anti-mouse FITC-conjugated secondary antibodies (dilution 1:500; DAKO, Glostrup, Denmark) for 20 minutes in the dark. The signal from labelled cells was acquired using a FACSCalibur and was analysed with FlowJo (Tree Star, Ashland, OR, USA), following the manufacturer’s protocol, with gating selected at the point of intersection between the marker and its isotype control [38 (link)].
CM differentiation efficiency was quantified by the expression of myosin heavy chain (MHC) and cTnT I. Briefly, cells harvested from day 20 were fixed and permeabilised using Fix and Perm Cell permeabilisation reagents (Life Technologies). The cells were subsequently incubated with anti-MHC (dilution 5:200; Developmental Studies Hybridoma Bank, Iowa city, IA, USA) and anti-cTnT (dilution 1:200; Millipore) for 20 minutes. After washing with 1% bovine serum albumin/PBS, the cells were incubated in the dark with 1:500 dilutions of anti-mouse FITC-conjugated secondary antibodies (dilution 1:500; DAKO, Glostrup, Denmark) for 20 minutes in the dark. The signal from labelled cells was acquired using a FACSCalibur and was analysed with FlowJo (Tree Star, Ashland, OR, USA), following the manufacturer’s protocol, with gating selected at the point of intersection between the marker and its isotype control [38 (link)].
4
Surface Biotinylation and Western Blot Analysis
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Surface biotinylation was carried out as explained earlier. [54 (link)]. In brief, C2C12 myoblasts were cultured for various differentiation times, after which the surface proteins were biotinylated by exposure to NHS-LC-biotin (Thermo) in PBS for 30min at 4°C. After quenching with 100mM glycine, cells were lysed in lysis buffer (10mM Tris-HCl pH7.2, 150mM NaCl, 1% Triton X-100, 1mM EDTA) with proteinase inhibitor (Roche Diagnostics) for 1hr at 4°C. Biotinylated proteins were collected from streptavidine-agarose beads (Pierce), then the samples were analysed by SDS-PAGE and PVDF-membrane transfer.
Western blot analysis was conducted as explained earlier [40 (link)]. In brief, cell lysis was performed with the lysis buffer composed of 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and then, the prepared samples were separated by SDS-PAGE. The antibodies used were as follows. Anti-Kir2.1 (1:500), anti-Myogenin (1:500), anti-β-tubulin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pan-Cadherin (1:1000) (Sigma-Aldrich, St Louis, MO), anti-CDO (1:500) (R&D Systems, Minneapolis, MN) and anti-MHC (1:500) (MF20: Developmental Studies Hybridoma Bank, Iowa, IA).
Western blot analysis was conducted as explained earlier [40 (link)]. In brief, cell lysis was performed with the lysis buffer composed of 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and then, the prepared samples were separated by SDS-PAGE. The antibodies used were as follows. Anti-Kir2.1 (1:500), anti-Myogenin (1:500), anti-β-tubulin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pan-Cadherin (1:1000) (Sigma-Aldrich, St Louis, MO), anti-CDO (1:500) (R&D Systems, Minneapolis, MN) and anti-MHC (1:500) (MF20: Developmental Studies Hybridoma Bank, Iowa, IA).
5
Comprehensive Protein Analysis via Western Blotting
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Western blotting was performed, as previously described (Morita and Hayashi, 2018 (link)). Briefly, total proteins were extracted from cells with 2% sodium dodecyl sulfate (SDS) sample buffer. The proteins were electrophoretically separated using 10% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. For western blotting, anti-Arp5 (Proteintech), anti-MyoG (Santa Cruz Biotechnology, Inc), anti-MyoD (Santa Cruz Biotechnology, Inc), anti-MYF6 (Santa Cruz Biotechnology, Inc), anti-MHC (Developmental Studies Hybridoma Bank), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Thermo Fisher Scientific), anti-FLAG (Sigma-Aldrich), anti-HA (Roche Applied Science), and anti-Myc (Santa Cruz Biotechnology, Inc) antibodies were used as primary antibodies. The raw images of western blotting can be found in the source data files.
6
Protein Expression Analysis in CFM Cells
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The CFM cells and chicken tissues were lysed in lysis buffer (50 mM Tris⁄HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40, 50 mM NaF, 1mM Na3VO4, 5mM β-glycerophosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). Equal amounts of total protein were separated via 12% SDS-PAGE, transferred to a PVDF membrane, and probed with anti-chicken CARP, anti-MHC (MH-20, Developmental Studies Hybridoma Bank), anti-CyclinD1 (Cloud-clone Corp), anti-p21 (GeneTex), anti-p27 (Novus Biologicals), anti-Cavolin-3 (Abcam), anti-MyoD (LSBio) anti-myc (CellBiolabs) or an anti-actin antibody (Santa Cruz). The detected proteins were visualized with the ECL detection system (Amersham Biosciences).
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