Freshly isolated spinal cord tissue was washed in pre-cooled PBS, and put into the RIPA lysis solution. The mixture was thoroughly ground and decomposed on ice for 30 minutes, followed by centrifugation to obtain supernatant. The protein in the supernatant was separated by polyacrylamide gel electrophoresis, and the separated protein was transferred to the PVDF membrane. The protein-containing PVDF membrane was incubated with 5% nonfat dried milk for 1 hour, and then incubated with anti-rabbit P2Y14 (1:500, APR026, Alomone Labs), IL-1β (1:500, AF5103, A nity), TNF-α (1:500, PB0082, Boster), p38 MAPK (1:800, 8690, Cell Signaling Technology), p-p38 MAPK (1:800, 4511, Cell Signaling Technology) overnight at 4°C. After three washes with TBST, the membranes were incubated in secondary antibody with horseradish peroxidase (1:2000, Beijing Zhongshan Biotechnology Co.) for 1 h. Put the membranes into the gel imaging system and the ECL luminescent solution was added to expose the image. The results were recorded and stored. The optical density analysis of protein results was analyzed by Image-Pro Plus software, and the expression of target protein in each group was standardized by β-actin.
Horseradish peroxidase
Manufactured by Zhongshan Biotechnology
Sourced in United Kingdom
Horseradish peroxidase is an enzyme isolated from the horseradish plant. It catalyzes the oxidation of various substrates in the presence of hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
P38 mapk, by Cell Signaling Technology (2 mentions)
P p38 mapk, by Cell Signaling Technology (2 mentions)
Pb0082, by Boster Bio (2 mentions)
Apr026, by Alomone (2 mentions)
Ripa kit, by Solarbio (1 mentions)
Ab19857, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab92494, by Abcam (1 mentions)
Ab19898, by Abcam (1 mentions)
Ab40866, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Ecl808 25, by Biomiga (1 mentions)
3 protocols using horseradish peroxidase
1
Western Blot Analysis of Spinal Cord Proteins
2
Western Blot Analysis of Spinal Cord Proteins
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Freshly isolated spinal cord tissue was washed in pre-cooled PBS, and put into the RIPA lysis solution. The mixture was thoroughly ground and decomposed on ice for 30 minutes, followed by centrifugation to obtain supernatant. The protein in the supernatant was separated by polyacrylamide gel electrophoresis, and the separated protein was transferred to the PVDF membrane. The protein-containing PVDF membrane was incubated with 5% nonfat dried milk for 1 hour, and then incubated with anti-rabbit P2Y14 (1:500, APR026, Alomone Labs), IL-1β (1:500, AF5103, A nity), TNF-α (1:500, PB0082, Boster), p38 MAPK (1:800, 8690, Cell Signaling Technology), p-p38 MAPK (1:800, 4511, Cell Signaling Technology) overnight at 4°C. After three washes with TBST, the membranes were incubated in secondary antibody with horseradish peroxidase (1:2000, Beijing Zhongshan Biotechnology Co.) for 1 h. Put the membranes into the gel imaging system and the ECL luminescent solution was added to expose the image. The results were recorded and stored. The optical density analysis of protein results was analyzed by Image-Pro Plus software, and the expression of target protein in each group was standardized by β-actin.
3
Protein Expression Analysis in Cells
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Total protein was extracted from the tissues and cells using a radioimmunoprecipitation assay (RIPA) kit (R0010, Solarbio Technology, Beijing, China). After being separated by PAGE, the protein was transferred to a nitrocellulose membrane and blocked with 5% BSA for 1 h at room temperature. The membrane was incubated with diluted rabbit monoclonal antibodies specific for CHEK1 (ab40866, Abcam, Cambridge, UK), CD133 (ab19898, Abcam, Cambridge, UK), OCT4 (ab19857, Abcam, Cambridge, UK), SOX2 (ab92494, Abcam, Cambridge, UK), Bax (ab32503, Abcam, Cambridge, UK), and Bcl-2 (ab32124, Abcam, Cambridge, UK) at 4°C for 12 h. The membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibodies (Zhongshan Biotechnology, Beijing, China, diluted at 1:5,000) and then reacted with enhanced chemiluminescence solution (ECL808-25, Biomiga, San Diego, CA, USA) for 1 min at room temperature. X-ray images (36209ES01, Qianchen Biological Technology, Shanghai, China) were acquired. Using β-actin as the internal reference, the ratio of the gray value of the target band to the inner reference band was used as the relative expression of the protein. Each experiment was repeated three times.
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