Cells were digested with low-concentration trypsin and collected into tubes. We used a Nuclear and Cytoplasm Protein Extraction Kit (Beyotime) to separate nuclear and cytoplasm proteins. Cells were lysed in an ice cold radio immunoprecipitation assay (RIPA) lysis buffer with 1 mM phenylmethyl sulfonyl fluoride (Sigma-Aldrich), which was used for cell protein extraction. Protein concentration was determined using a BCA KIT (Beyotime). Proteins were separated by 4%–12% SurePAGE gels (GenScript, Nanjing, China), transferred to a nitrocellulose membrane (Pall, Mexico), and then detected using antibodies according to standard procedures. Primary antibodies were applied overnight at 4 °C for western blot tests, and their dilutions were as follows: NCAPG (sc-101014, Santa Cruz, 1:1000); MYHC (MF20, Developmental Studies Hybridoma Bank, 1:50); MYOD (sc-377460, Santa Cruz, 1:1000); MYOG (sc-12732, Santa Cruz, 1:1000) and β-tubulin (10094-1-AP, Proteintech, 1:2000). Finally, secondary antibodies were visualized with HRP-conjugated secondary antibodies that were applied for 1 h at room temperature. ECL western blotting detection reagent (Beyotime) was used to visualize the protein bands.
Myog sc 12732
Manufactured by Santa Cruz Biotechnology
Sourced in United States
MyoG (sc-12732) is a protein-specific antibody developed by Santa Cruz Biotechnology for use in research applications. It is designed to detect the presence and quantity of the MyoG protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mef2c, by Santa Cruz Biotechnology (3 mentions)
Tritc labeled secondary antibody, by Jackson ImmunoResearch (3 mentions)
Surepage gel, by GenScript (1 mentions)
Nuclear and cytoplasm protein extraction kit, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Nitrocellulose membrane, by GenScript (1 mentions)
Ecl western blotting detection reagent, by Beyotime (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab83832, by Abcam (1 mentions)
Sc 376157, by Santa Cruz Biotechnology (1 mentions)
Sab4501982, by Merck Group (1 mentions)
Ab2722, by Abcam (1 mentions)
Ab2796, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
Sc 20641, by Santa Cruz Biotechnology (1 mentions)
5 protocols using myog sc 12732
1
Nuclear and Cytoplasmic Protein Extraction
2
Quantifying Myogenic Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After myoblasts were treated with differentiation medium (DM) containing HG-DMEM supplemented with 2% horse serum (HS, Sigma, USA) for the indicated time, the differentiated myoblasts were stained for MyoG or MEF2C using the primary polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruz) or MEF2C (5030S, 1:400, CST) and the appropriate TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were stained with DAPI. C2C12 myoblasts with only 1–2 nuclei within a cellular structure were evaluated with MyoG or MEF2C staining. MyoG + or MEF2C + cells were defined as differentiated cells that did not fuse to form myotubes. Myoblasts with 3 or more nuclei in the structure of a cell were defined as myotubes. The number of double-positive nuclei in a high-power field (HPF, 50 μm) was analyzed after double staining with MyoG/DAPI or MEF2C/DAPI. Two individuals who were blinded to the results evaluated the images using ImageJ (Java) software (National Institutes of Health, USA).
3
Quantifying Myogenic Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After myoblast cells were treated under DM for the indicated time, the differentiated myoblast cells were stained for MyoG or MEF2C using the primary polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruze) or MEF2C (5030S, 1:400, CST) and appropriative TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were stained with DAPI. C2C12 myoblast cells with only 1-2 nucleuseswithin a cellular structure were evaluated withMyoG or MEF2C staining.TheMyoG + or MEF2C + cells were de ned as the differentiated cells that did not fuse to form myotubes. Myoblast cells with 3 or morenucleusesin the structure of a cell were de ned as myotubes. The number of double-positive nuclei under high power eld (HPF, 50 µm) were analyzed afterdouble staining of MyoG/DAPI or MEF2C/DAPI. Two individuals who did not know the results evaluated the images using Image J (Java) software (National Institutes of Health, USA).
4
Quantifying Myogenic Differentiation Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After myoblast were treated under DM for the indicated time, the differentiated myoblast was stained for MyoG or MEF2C using the primary polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruze) or MEF2C (5030S, 1:400, CST) and appropriative TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were stained with DAPI. C2C12 myoblast with only 1-2 nuclei within a cellular structure were evaluated with MyoG or MEF2C staining. The MyoG+ or MEF2C+ cells were de ned as the differentiated cells that did not fuse to form myotubes. Myoblast with 3 or more nucleiin the structure of a cell were de ned as myotubes. The number of double-positive nuclei under high power eld (HPF, 50 μm) were analyzed after double staining of MyoG/DAPI or MEF2C/DAPI. Two individuals who did not know the results evaluated the images using Image J (Java) software (National Institutes of Health, USA).
5
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2C12 myoblastwere homogenized on ice in 0.1% Tween-20 homogenization buffer containing protease inhibitors. Nuclear and cytosolic proteins were separated and collected using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacture's instruction (78835, Thermo Fisher Scienti c, USA). 20 µg of protein in each well were separated by 7 or 10 % SDS-PAGE gel electrophoresis and transferred onto PVDF membrane (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies, including α-tubulin (T9026, 1:5000, Sigma), Histone H3 (ab6002, 1:500, ABCAM), MyoG (sc-12732, 1:250, Santa Cruze), MEF2C (5030S, 1:500, CST), NFATc1 (ab2796, 1:500, Abcam), NFATc2 (ab2722,1:500, Abcam), NFATc3(ab83832,1:500, Abcam),NFATc4 (SAB4501982, 1:1000, Sigma) and MyHC (sc-20641, sc-376157, 1:500, Santa Cruze) overnight at 4 °C, respectively. Thereafter, the blots were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, anti-goat IgG, 1:10000; Santa Cruz) for 90 min. Protein expression was detected by enhanced chemiluminescence method, and the Image J software was used for gray value analysis 19 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!