Total heart ventricle lysate was prepared in radio-immunoprecipitation assay (RIPA) buffer containing freshly added cocktail protease inhibitors and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). Protein lysates were subjected to SDS-PAGE as described [47 (link),48 (link)]. Filters were sequentially incubated with primary antibodies, then horseradish-peroxidase-conjugated species-specific secondary antibodies (Bio-Rad, Hercules, CA, USA), followed by enhanced chemiluminescence reaction (Bio-Rad Laboratories Inc., Hercules, CA, USA). Densitometric analyses were performed with Image J software.
Horseradish peroxidase conjugated species specific secondary antibodies
Manufactured by Bio-Rad
Sourced in United States
Horseradish-peroxidase-conjugated species-specific secondary antibodies are specialized laboratory reagents used for immunodetection techniques. They consist of secondary antibodies that are chemically conjugated to the enzyme horseradish peroxidase. These conjugated antibodies can be used to detect and visualize target proteins in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence reaction, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
Nupage gel, by Thermo Fisher Scientific (2 mentions)
Biomax mr film, by Kodak (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Enhanced chemiluminescence substrate, by Bio-Rad (1 mentions)
Edta free protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using horseradish peroxidase conjugated species specific secondary antibodies
1
Western Blot Analysis of Total Heart Lysate
2
Protein Extraction and Western Blotting
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Cells were lysed in radioimmunoprecipitation assay buffer containing freshly added cocktail protease inhibitors [complete, ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail; Sigma-Aldrich, St. Louis, MO]. Western Blots were performed as previously described (Cozzolino et al., 2013 (link)) using the following primary antibodies: rabbit polyclonal α-HNF1α (NBP1-33596, 1:1000; Novus Biologicals, USA), rabbit polyclonal α-CBP/p300 (451, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit monoclonal α-cyclin-dependent kinase 4 (CD22, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and mouse monoclonal α-glyceraldehyde 3-phosphate dehydrogenase (MAB374, 1:1000; Millipore Corp., Bedford, MA, USA). Blots were then incubated with horseradish-peroxidase-conjugated species-specific secondary antibodies (Bio-Rad, Hercules, CA, USA), followed by enhanced chemiluminescence reaction (Bio-Rad Laboratories Inc., Hercules, CA, USA). Densitometric analyses were performed with ImageJ.
3
Western Blot Analysis of Kidney Proteins
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Total kidney lysates covering all kidney zones were prepared in radioimmunoprecipitation assay (RIPA) buffer containing freshly added cocktail protease inhibitors [complete, ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail; Sigma-Aldrich, St. Louis, MO]. Cell lysates were also prepared with RIPA mentioned above. Protein lysates were subjected to SDS-PAGE as described previously (Li et al., 2020 (link); Shi et al., 2020 (link)) and transferred to polyvinylidene fluoride membranes. After incubation with primary antibodies, the filters were incubated with horseradish-peroxidase conjugated species-specific secondary antibodies (Bio-Rad, Hercules, CA), followed by application of enhanced chemiluminescence substrate (Bio-Rad Laboratories Inc., Hercules, CA). Densitometric analyses were performed with Image J software.
4
Western Blot Analysis of Protein Samples
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Cell lysates were separated under reducing conditions on NuPAGE gels (Life Technologies). Proteins were transferred to nitrocellulose and blocked in 5% (w/v) nonfat milk in PBS containing 0.2% Tween 20. Primary antibodies were diluted in 5% (w/v) nonfat milk in PBS containing 0.2% Tween 20. Species-specific horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) were used, and the signal was developed using enhanced chemiluminescence (GE Healthcare). Blots were exposed to Biomax MR film (Eastman Kodak Co.), and images were prepared for publication using Adobe Photoshop.
5
Western Blot Analysis of Protein Samples
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Cell lysates were separated under reducing conditions on NuPAGE gels (Life Technologies, Inc.). Proteins were transferred to nitrocellulose and blocked in 5% w/v nonfat milk in PBS + 0.2% Tween 20. Primary antibodies were diluted in 5% w/v nonfat milk in PBS + 0.2% Tween 20. Species-specific horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) were used, and the signal was developed using enhanced chemiluminescence (GE Healthcare). Blots were exposed to BiomaxMR film (Eastman Kodak), and images were prepared for publication using Adobe Photoshop.
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