Equal protein loading was prepared before resuspending with 2 × Laemmli sample buffer. The mixture was collected by short spin centrifugation and boiled for 5 min before loaded into SDS–polyacrylamide gel electrophoresis. Lysate was separated on SDS–polyacrylamide gel electrophoresis (6, 8, or 15% acrylamide) with 200 V for 1 h. The separated protein was transferred onto the nitrocellulose membrane using the Transblot Turbo® Transfer System (BioRad, UK). The membranes were blocked in 5% reduced-fat milk for 1 h before incubate with primary antibodies. The antibodies are Fast-MyHC, α-tubulin, SSRP-1 (1:1000) and Desmin, (1:2000), from Sigma-Aldrich, UK. PTEN (1:1000) (a gift from Dr. Zubair Ahmed, Medical School, University of Birmingham). PI3K, phospho-PI3K (Tyr458), Akt, phospho-Akt (Ser473), mTOR, phospho-mTOR (Ser2448), p70S6 Kinase, phospho-p70S6 Kinase, Rictor, phospho-Rictor (Thr1135) and FoxO3a (1:1000) from Cell Signalling Technology, UK. Beclin1, Atg5, Atg7, and LC3B (1:1000) (a gift from Dr. Melissa Grant, School of Dentistry, University of Birmingham).
Fast myhc
Manufactured by Merck Group
Sourced in United Kingdom
Fast-MyHC is a laboratory equipment product designed for the detection and analysis of fast myosin heavy chain (MyHC) isoforms. It provides a reliable and efficient means to measure the expression levels of fast MyHC subtypes in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Merck Group (1 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Desmin, by Merck Group (1 mentions)
Phospho p70 s6 kinase, by Cell Signaling Technology (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Phospho rictor thr1135, by Cell Signaling Technology (1 mentions)
Foxo3a, by Cell Signaling Technology (1 mentions)
Rictor, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg fitc, by Bioss Antibodies (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Ibitreat slide, by Ibidi (1 mentions)
Desmin, by Abcam (1 mentions)
Myogenin, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
4 protocols using fast myhc
1
Western Blotting Protocol for Protein Analysis
2
Quantification of Myosin Heavy Chain Isoforms
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Cells were collected from 6-well plates, washed twice with PBS, and then lysed in 150 μl radioimmunoprecipitation assay buffer containing 1% protease inhibitor (Beyotime; catalog no.: P0013B). Western blotting was performed according to a previously established procedure (28 (link)). The primary antibodies targeted the following proteins: MyHC I (1:1000 dilution; DSHB), MyHC IIA (1:1000 dilution; DSHB), MyHC IIX (1:1000 dilution; DSHB), MyHC IIB (1:1000 dilution; DSHB), Fast-MyHC (1:1000 dilution; Sigma), Slow-MyHC (1:1000 dilution; Sigma), and β-tubulin (1:200 dilution; Santa Cruz Biotechnology). The secondary antibody is antimouse horseradish peroxidase (1:5000 dilution; Santa Cruz Biotechnology).
3
Immunofluorescence Staining of Fast-MyHC in Cells
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The cells were washed with PBS twice and fixed with 4% paraformaldehyde (Biosharp) solution for 10 to 15 min at room temperature and then washed with PBS for three times. Subsequently, cells were incubated with 100 nM glycine for 10 min, washed with PBS three times, and then blocked with blocking buffer (5% goat serum, 2% bovine serum albumin, 0.2% Triton X-100, and 0.1% sodium azide) for 30 min at room temperature. Cells were immunized with primary antibody (fast-MyHC, 1:1000 dilution, catalog no.: M4276; Sigma) at 4 °C overnight, and fluorescence-labeled secondary antibody (goat antimouse IgG/FITC; Bioss) at room temperature for 1 h. 4′,6-Diamidino-2-phenylindole (1:1000 dilution, catalog no.: D1306; Thermo Scientific) was then used for nuclear staining. Finally, photographs were taken using a fluorescent inverted microscope (Olympus). Five views per photo were selected and counted, and the average was set as the value of this sample for further statistical analysis.
4
Immunofluorescence analysis of induced myogenic cells
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Induced myogenic cells (iMCs) grown on Matrigel-coated 8-well IbiTreat slides (Ibidi) were fixed with 3.7% Formaldehyde (10 min). Cells were permeabilized with 0.1% Tween 20 in TBS and blocked in 0.1% Triton X-100 + 1% FBS in TBS for 30 min. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C on a shaker (Desmin (1:1000, Abcam ab15200), fast MyHC (1:200, Sigma-Aldrich M4276), MyHC3 (1:200, Santa-Cruz sc-2064), Myogenin (1:800, Abcam ab1835), PAX7 (1:200, Santa-Cruz sc-81648), TUJ1 (1:1000, Sigma-Aldrich T8578)). Next day, secondary antibodies were also diluted in blocking solution and again incubated overnight at 4 °C on a shaker (AlexaFluor 568 goat anti-mouse (1:1000, Thermo Fisher A11031), AlexaFluor 568 donkey anti-rabbit (1:1000, Thermo Fisher A10042), AlexaFluor 488 goat anti-mouse (1:1000, Thermo Fisher A11001)). Hoechst (1:10,000 in DPBS) was incubated for 5 min at RT. Confocal immunofluorescence imaging was performed using the Laser Scan Microscope LSM 700 (Carl Zeiss, Jena, Germany) and for mosaic image acquisition a Leica DMI 6000 B microscope equipped with a XY scanning stage (Leica Microsystems) was used.
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