We followed the protocols described in our studies (Yao and Abdel-Rahman, 2016 (link), Ibrahim et al., 2014 (link), Yao and Abdel-Rahman, 2017 (link)). The supernatant (40 µg of protein) of the homogenized LV tissue was separated in a 4–12% gel electrophoresis (Novex Tis-Glycine gel, Life Technologies, CA), and transferred to nitrocellulose membrane, and then incubated with a primary antibody (mouse anti-p-ERK1/2 and rabbit anti ERK1/2; 1:500, Cell Signaling, Danvers, MA). Thereafter, the membrane was incubated with a secondary antibody (IRDye680-conjugated goat anti-mouse and IRDye800-conjugated goat anti-rabbit, 1:15000, LI-COR Biosciences, Lincoln, NE), and the protein bands were detected and quantified by Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, NE).
Protocol detail
Quantification of Phosphorylated ERK1/2 in LV Tissue
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Novex Tis-Glycine gel
mouse anti-p-ERK1/2
rabbit anti ERK1/2
IRDye680-conjugated goat anti-mouse
IRDye800-conjugated goat anti-rabbit
Odyssey Infrared Imager
Antibody
Electrophoresis
Erk1
Glycine
Goat
Irdye800
Membrane
Mouse
Nitrocellulose
Protein
Rabbit
Tissue
Corresponding Organization : East Carolina University
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Variable analysis
independent variables
- Supernatant (40 µg of protein) of the homogenized LV tissue
- Levels of phosphorylated ERK1/2 (p-ERK1/2)
- Levels of total ERK1/2
- Gel electrophoresis (4–12% gel)
- Nitrocellulose membrane for protein transfer
- Primary antibodies (mouse anti-p-ERK1/2 and rabbit anti ERK1/2; 1:500)
- Secondary antibodies (IRDye680-conjugated goat anti-mouse and IRDye800-conjugated goat anti-rabbit; 1:15000)
- Odyssey Infrared Imager for protein band detection and quantification
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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