The protein samples were loaded onto an 8% or 11% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) system for electrophoresis and blotting. The following antibodies were used: rabbit anti-ABCA1, anti-ABCG1 antibody (Sangon Biotech, Shanghai, China), rabbit anti–NPR-A, anti-LXRα antibody (ABclonal Technology, Wuhan, China), and rabbit anti-PPARγ, and β-actin antibody (Wanleibio, Shenyang, China). After incubation with the horseradish peroxidase–conjugated secondary antibody, the protein bands were visualized by enhanced chemiluminescence (ECL) (Wanleibio, Shenyang, China).
Enhanced chemi luminescence (ecl)
Manufactured by Wanlei
Sourced in China
The ECL (Enhanced Chemiluminescence) is a laboratory equipment used for the detection and quantification of proteins in biological samples. It utilizes a chemiluminescent reaction to generate light, which is then measured and analyzed to provide information about the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ab179463, by Abcam (1 mentions)
Ab6702, by Abcam (1 mentions)
Ab154931, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab192623, by Abcam (1 mentions)
Ripa lysis buffer, by Wanlei (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Cep290, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
4 protocols using enhanced chemi luminescence (ecl)
1
Western Blot Analysis of ABCA1, ABCG1, NPR-A, LXRα, and PPARγ
2
Western Blot Analysis of Protein Expression
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RIPA lysis buffer (Wanlei Bio, China) containing protease inhibitor cocktail (Roche) was used to extract the protein from the harvested cells. Then, the protein samples were loaded onto 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% BSA, the PVDF membranes were incubated with primary antibodies overnight at 4°C and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Thereafter, enhanced chemiluminescence (ECL) (Wanlei Bio, China) was used to detect the protein bands. The following antibodies were included: GAPDH (WL01114, 1:2000 dilution, Wanlei Bio, China); TRIM27 (ab154931, 1:500 dilution, Abcam, USA); Akt (ab179463, 1:1000 dilution, Abcam, USA); p-Akt (ab192623, 1:1000 dilution, Abcam, USA); Goat Anti-mouse IgG H&L (HRP) (ab205719, 1:2000 dilution, Abcam, USA) and Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab6702, 1:2000 dilution, Abcam, USA).
3
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from cells, and protein levels were measured using a BCA protein assay kit (Beyotime, Shanghai, China). Proteins were separated by 8% SDS-PAGE and transferred to PVDF membranes. After blocking in 5% skimmed milk, the membranes were incubated with primary antibodies at 4° C overnight. Next day, the membranes were rinsed with TBST three times, incubated for 1 h with secondary antibody at ambient temperature, rinsed with TBST three times and visualized by ECL (Wanleibio, Shenyang, China). The following antibodies CEP290 (ab85728, Abcam, USA), Nrf2, NQO1, HO-1 (Proteintech, Wuhan, China), FTH1 (Cell Signaling Technology, USA) and β-actin (DianyinBio, Shanghai, China) were used in this study.
4
Quantitative Western Blot Analysis
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Protein was isolated from cells and subjected to sonication in ice-cold lysis buffer. Denatured protein was separated on sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). After blocking for 2 h at room temperature, the PVDF membrane was incubated with anti-Collagen Type VI [Proteintech, CHI, USA (17023-1-AP)], anti-MYCT1 [Abcam, Cambridge, UK (ab139945)], and anti-β-actin [Cloud-Clone Corp. USA (CAB340Mi22)] primary antibodies, followed by incubation with appropriate peroxidase-coupled secondary antibodies. Proteins on the PVDF membrane were stained with ECL (WanleiBio, Shenyang, China) and visualized using ChemiDocTM Touch Imaging System (Bio-Rad, CA, USA). The relative protein expression of each gene normalized to β-actin was calculated based on integrated band density by Image Lab software (Bio-Rad, CA, USA).
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