Lysates of mouse kidneys, hearts, livers and human hearts were prepared by homogenization in radioimmunoprecipitation assay (RIPA) buffer containing SDS (Santa Cruz Biotechnology) with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher). Lysates of mouse hippocampi were prepared as described above and the P2 fractions were run for western blotting. Cultured cells were lysed directly in M-PER mammalian protein extraction reagent (Thermo Fisher) supplemented with Halt protease and phosphatase inhibitor cocktail. Equal amounts of protein were loaded, and electrophoresis was performed in NuPAGE 4–12% gradient bis-tris polyacrylamide protein gels (Thermo Fisher). Proteins were transferred to PVDF membrane and blocked with 5% milk in phosphate-buffered saline with Tween-20 for 1 hour. Membranes were then incubated overnight with primary antibody at 4 °C. Blots were washed and incubated with secondary antibody for 1 hour at room temperature. After washing, the secondary antibody was visualized by Pierce ECL chemiluminescence reagents (Thermo Fisher) or using a LI-COR Odyssey imaging system (LI-COR Biosciences).
Pierce ecl chemiluminescence reagents
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The Pierce ECL chemiluminescence reagents are a set of reagents designed for the detection of proteins in Western blot analysis. These reagents produce a luminescent signal when they react with the horseradish peroxidase (HRP) enzyme, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nupage 4 to 12 gradient bis tris polyacrylamide protein gels, by Thermo Fisher Scientific (1 mentions)
Biorad wet transfer system, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated goat anti rabbit immunoglobulin g, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid protein assay kit, by Tiangen Biotech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Imagequant las 4000, by Thermo Fisher Scientific (1 mentions)
Tubulin antibody 11224 1 ap, by Proteintech (1 mentions)
Atg7 antibody, by Cell Signaling Technology (1 mentions)
Lc3 antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
4 protocols using pierce ecl chemiluminescence reagents
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of Frozen Samples
3
Western Blot Analysis of K-Ras Protein
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Protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. After blocking, the membranes were incubated with purified rabbit anti-K-Ras antisera at 4°C overnight. The next day, the membranes were washed with phosphate-buffered saline and then incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology, Texas, USA). Immunodetection was conducted with Pierce® ECL chemiluminescence reagents (Thermo Fisher) and exposed on an X-ray film. β-actin was used as an internal reference for relative quantification.
4
Western Blot Analysis of Autophagy Proteins
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The following antibodies were sued in this study: ATG7 antibody (#8558; Cell Signaling Technology), LC3 antibody (#3868; Cell Signaling Technology), BECN1 antibody (#3738; Cell Signaling Technology), tubulin antibody (11224-1-AP; Proteintech Group, Chicago, IL, USA); caspase-8 antibody (13423-1-AP; Proteintech Group); antirabbit IgG, horseradish peroxidase (HRP)-linked antibody (#7074; Cell Signaling Technology) and anti-mouse IgG, HRP-linked antibody (#7076; Cell Signaling Technology). Cells were lysed with the radioimmunoprecipitation assay (RIPA) buffer (P0013C; Beyotime Biotechnology), and the total protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (PA115; Tiangen Biotech). In addition, proteins from whole-cell extracts were resolved using denaturing SDS-PAGE and analyzed by Western blotting. Subsequently, equal amounts of protein were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). Protein bands were detected with Pierce ECL chemiluminescence reagents (Thermo Fisher) and exposed on the ImageQuant LAS 4000 (Japan) system. Immunoblots shown in figures were derived from three independent experiments. Furthermore, intensities of protein bands were quantified by densitometry using the ImageJ software (http://imagej. nih.gov/ij/).
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