The cortical tissue (30 mg) surrounding the lesioned areas were lysed in lysis buffer (Santa Cruz Biotechnology), and the total protein levels were quantified. A total of 25 μg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane that were blocked in 10% skimmed milk at room temperature for 2 h. The solution was incubated at 4°C overnight with primary antibodies against H3, H4, acetyl-H3, acetyl-H4, acetyl-Nrf2 (Cell Signaling Technology), cleaved caspase-3, LC3, Beclin, ATG-3 and ATG-7 (Cell Signaling Technology), Bax, ROS and GFAP (Abcam), Iba-1, Nrf2, HO-1, NQO1 and UGT1A1 (Santa Cruz Biotechnology Inc.), followed by incubation with appropriate secondary antibodies. Immunoblots were visualized using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3000, Cell Signaling Technology).
Millipore ecl western blotting detection system
Manufactured by Merck Group
Sourced in United States
The Millipore ECL Western Blotting Detection System is a laboratory equipment used for the detection and visualization of proteins in Western blot analysis. It employs a chemiluminescent detection method to amplify and detect the signal from labeled target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
Un scan it 6, by Silk Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
P erk erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Quantity one system, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
3 protocols using millipore ecl western blotting detection system
1
Western Blot Analysis of Protein Expression in Cortical Tissue
2
Western Blot Analysis of Protein Expression
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Total protein was isolated from C6 cells or primary human astrocytes using ice-cold RIPA buffer. Total protein concentrations were measured with the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein samples (30 μg per lane) were separated using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Proteins were detected by incubation with primary antibodies (p-Src/Src, p-ERK/ERK, NF-κB p65, p-NF-κB p65, histone H3, or GAPDH at 1:1000 from Cell Signaling, Danvers, MA, USA; or σ-1R at 1:500 from Invitrogen, Carlsbad, USA) followed by secondary antibodies (horseradish peroxidase-conjugated to goat anti-mouse/rabbit IgG at 1:2,000). Glial fibrillary acidic protein (GFAP) and β-actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA) were employed as loading controls. Immunoblots were visualized using Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Signals were detected by chemiluminescence and imaged on the Microchemi 4.2 (DNR, Israel) digital image scanner. Quantification was performed by densitometry using Image J software (NIH).
3
Western Blot Analysis of Liver Proteins
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The total proteins in the liver tissues were extracted using RIPA reagents (Applygen, Beijing, China) supplemented with phosphatase inhibitors and protease (Beyotime, Shanghai, China). Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membranes were incubated overnight with primary antibodies (1:1000). Antibodies of NLRP3, caspase-1, and GAPDH were purchased from Proteintech Technology (Wuhan, China). The membranes were then incubated for 2 h with the secondary antibody (1:5000; Cell Signaling Technology, Beverly, USA). The bands were exposed using a Millipore ECL Western Blotting Detection System (Millipore, Billerica, USA) and were quantified using the Quantity One System (Bio-Rad, Hercules, USA).
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