Protein lysates were obtained by homogenization of mice cortex, lumbar spinal cord, and hypothalamus in homogenization buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100, 10 mM EDTA) added with protease inhibitor cocktail (Sigma-Aldrich). After sonication, lysates were kept on ice and centrifuged for 20 min at 14,000 × g at 4°C. Primary microglia were harvested in ice-cold RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) added with protease inhibitor cocktail (Sigma-Aldrich). Lysates were kept on ice and then centrifuged for 10 min at 14,000 × g at 4°C. Supernatants were assayed for protein quantification with the Bradford detection kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by SDS-PAGE gels and transferred onto a nitrocellulose membrane (Amersham Biosciences, Cologno Monzese, Italy). Membranes were incubated with the specified antibodies overnight at 4°C and with HRP-conjugated secondary antibodies for 1 h and then detected using ECL Advance WB detection kit (Amersham Biosciences). Quantifications were performed by Kodak Image Station 440CF.
Bradford detection kit
Manufactured by Bio-Rad
Sourced in United States
The Bradford detection kit is a colorimetric assay used for the quantitative determination of protein concentrations. It is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Ecl advance wb detection kit, by Cytiva (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Mini protean tgxtm gel, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ecl advance detection kit, by Cytiva (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Ecl advance western blot detection kit, by Cytiva (1 mentions)
4 protocols using bradford detection kit
1
Extraction and Analysis of Protein Lysates
2
Gastrocnemius and Tibialis Anterior Muscle Protein Analysis
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Total protein extracts from mice gastrocnemius and tibialis anterior muscles were obtained in homogenization buffer (15 mg of dry tissue/150 μl of 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100, 10 mM EDTA) added with protease inhibitor cocktail (Sigma Aldrich). After sonication and centrifugation at 14000 × g (20 min at 4°C) supernatants were collected and assayed for protein content by Bradford detection kit (Bio-Rad Laboratories, Hercules, United States). Protein separation and analysis (15 μg/well) was performed by Mini-PROTEAN® TGXTM Gels (BioRad, United States) and by transfer onto nitrocellulose membranes. After saturation with 5% non-fat dry milk (1 h at room temperature), membranes were probed with gp91phox antibody (1:1000, BD Transduction Laboratories, United States) in 5% non-fat dry milk overnight at 4°C, and incubated with HRP-conjugated secondary antibody (mouse 1:5000, Jackson Immunoresearch) for 1 h at room temperature. Detections were performed on X-ray film (Aurogene, United States), using ECL Advance detection kit (Amersham Biosciences, United States) and signal intensity visualized by Kodak Image Station and analyzed by ImageJ software (NIH, United States). Values were normalized with mouse anti-GAPDH (1:2500, Sigma-Aldrich, Italy).
3
Western Blot Analysis of Protein Lysates
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Protein lysates collected in RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) were centrifuged for 20 min at 14,000× g at 4 °C. Supernatants were assayed for protein quantification with the Bradford detection kit (Bio-Rad Laboratories). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). Membranes were blocked in 5% non-fat dry milk and then incubated overnight at 4 °C with the indicated primary antibodies. After rinsing with Tris-buffered saline solution with 0.1% Tween-20 (TBS-T), membranes were incubated for 1 h with the appropriate peroxidase-conjugated secondary antibody, then washed and developed using the ECL chemiluminescence detection system (Roche) or Advance Western blot detection kit (Amersham Biosciences, Buckinghamshire, UK). Densitometric analyses were performed using the ImageJ software program (National Institutes of Health, Bethesda, MD, USA).
4
Protein Extraction and Analysis
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Cells in serum-free medium were harvested in ice-cold RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) added with protease inhibitor cocktail (Sigma Aldrich). Lysates were kept for 30 min in ice and then centrifuged for 10 min at 14,000× g at 4 °C. Protein lysates were also obtained by mice lumbar spinal cords segments and cortex in homogenization buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100, 10 mM EDTA) added with protease inhibitor cocktail (Sigma Aldrich). After sonication, lysates were kept for 30 min in ice and then centrifuged for 20 min at 14000× g at 4 °C. Supernatants were collected and assayed for protein content by the Bradford detection kit (Bio-Rad Laboratories, Hercules, CA, USA.). Analysis of protein components was performed by Mini-PROTEAN® TGX™ Gels (BioRad) and transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). After saturation with blocking agent, blots were probed overnight at 4 °C with the specified antibody, and then incubated for 1 h with HRP-conjugated secondary antibodies and visualized using ECL Advance Western blot detection kit (Amersham Biosciences). Signal intensity quantification was performed by Kodak Image Station analysis software. Values were normalized with mouse anti-GAPDH (1:2500, Calbiochem).
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