Notes & TipsDetecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide
Section snippets
Acknowledgments
The authors acknowledge funding by the Canadian Space Agency (9F007-052237/001/SR) and the Natural Sciences and Engineering Research Foundation, a postdoctoral fellowship from Foreign Affairs and International Trade Canada (W.M.), and assistance from W. Snow and S. Lakhi.
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Inactivation of horseradish peroxidase with crotonic acid for reprobing of western blotting
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2018, Free Radical Biology and MedicineCitation Excerpt :The percentage of lipid in photographs of 10 non-overlapping fields of liver (200X) was determined from digital images using NIH Image J to identify the average proportion of red pixels per field in each animal. Protein was extracted from muscle samples and assayed for protein concentration against a standard curve before gel electrophoresis and western blotting, to quantify the levels of atrogin-1, myostatin, and MyHCs relative to β-actin as previously reported [15,25,27,49]. Primary antibodies were: rabbit polyclonal anti-atrogin-1 (Fbx32, Santa Cruz, sc33782, 1:1000); rabbit polyclonal anti-myostatin (GDF8, Santa Cruz, sc6885-R, 1:100); anti-MyHC1 (Developmental Studies Hybridoma Bank (DSHB), BA-D5-s, 1:1000); anti-MyHC2a (DSHB, SC-71-s, 1:750); anti-MyHC2b (DHSB, BF-F3-s, 1:10,000); and mouse anti-β-actin (Santa Cruz, 1:1500).
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2014, Toxicology LettersCitation Excerpt :The immunoreactive protein bands were detected with an Enhanced Chemiluminescence Luminal reagent (Keygen, Nanjing, China). The relative amount of protein was quantified from the relative optical density of the band by an image analysis system (Bio-Rad Gel Doc 2000, USA) (Upadhaya et al., 2011). All Western blotting experiments were repeated for six times.
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These authors contributed equally to the work.