The expression of Bax, Bcl-2, and caspase-3 was detected by Western blot. PQ-treated RAW264.7 cells were harvested and lysed in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and protease inhibitor cocktail tablets [Roche]) on ice. The protein concentrations of the cytosolic extracts were measured using the BCA protein assay with bovine serum albumin as standard. Equal amounts of protein extracts were separated on acrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a PVDF membrane and incubated with specific primary antibodies (rabbit polyclonal anti-Bcl-2, rabbit polyclonal anti-Bax, rabbit polyclonal anti-Caspase-3) overnight at 4°C. Each membrane was washed three times, and then incubated with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibodies for 1 h at room temperature. To prove equal loading, the blots were analyzed for β-actin expression using an anti-β-actin antibody. Immunodetection was performed using AmershamTM ECLTM western blotting detection reagents (GE Healthcare, Chalfont St. Giles, UK). Expression levels were quantified by densitometry, followed by normalization to the β-actin control. The signals were analyzed and quantified by Image J software (NIH, Bethesda, MD, USA).
Amersham ecltm
Manufactured by GE Healthcare
Sourced in United Kingdom
Amersham ECL is a chemiluminescent detection system used in Western blotting applications to detect and quantify proteins. It utilizes a light-emitting chemical reaction to visualize and analyze specific proteins in a sample.
Automatically generated - may contain errors
3 protocols using amersham ecltm
1
Apoptosis Signaling Pathway Analysis
2
Western Blot Protein Detection
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Western blotting assay was performed according to the previous studies (Zhong et al., 2012b (link); Quan et al., 2015 (link)). Briefly, cells were harvested and the total proteins were extracted with RIPA lysis buffer after the required treatments. Equal amounts of total proteins were separated by appropriate SDS-PAGE followed by transferring onto a PVDF membrane. After blocking with non-fat milk, the membrane was incubated with specific primary antibodies and the corresponding second antibodies, respectively. The specific protein bands were visualized with an AmershamTM ECLTM advanced western blotting detection kit (GE Healthcare Life Sciences, United Kingdom).
3
Western Blot Analysis of Adipose Tissue
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EVAT of Adipo-MROE and control mice were lysed for Western blotting as previously described [17 (link)]. Western blot samples were prepared with Laemmli Buffer (BioRad, Marnes-la-Coquette, France) and Criterion TGX Precast Gels (BioRad) were loaded with 30 µg of protein per well. Transfer was done on nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (BioRad). Membranes were incubated overnight with primary antibodies (1:1000 in Bovine Serum Albumin 3%, 4 °C), followed by 2 h with horseradish peroxidase-conjugated secondary antibodies (1:4000 in 5% skimmed milk, room temperature). Antibodies used are listed in Table S3 . Signals were detected by chemiluminescence (AmershamTM ECLTM, GE Healthcare, Buc, France), followed by signal acquisition with the ImageQuantTM LAS 4000 (Fujifilm, Tokyo, Japan). Blots were analysed densitometrically with ImageJ (ImageJ 1.43u). Quantification of phosphorylated proteins was normalised over total protein amount. β-actin was used as a housekeeping protein (1:4000 in Bovine Serum Albumin 3%, 4 °C), or the mitochondrial protein COX4 for TOM20 quantification to prevent variations due to mitochondrial mass. All reagents, otherwise indicated, were purchased from Sigma-Aldrich, Saint-Quentin-Fallavier, France.
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