Microglial cells were seeded in 6-well plates (3 × 105 cells/well) until 70–80% confluence was achieved. Protein extraction from tissues and cells was performed as described [42 (link)]. Briefly, proteins from BV2 cells were extracted by radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl 1% sodium deoxycholate, 1% Tryton X-100, 2 mM PMSF) (Sigma-Aldrich, Italy). After homogenization, the samples were spun at 12,000× g for 30 min, 4 °C, the supernatants were collected, and the insoluble pellet was separated. The total protein concentration was measured in a portion of each supernatant using Bradford colorimetric method (Sigma-Aldrich, Milan, Italy).
Bradford colorimetric method
Manufactured by Merck Group
Sourced in Germany
The Bradford colorimetric method is a laboratory technique used for the quantitative determination of total protein concentration in a sample. It is a widely used analytical method that relies on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a measurable color change that is proportional to the protein concentration.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by Merck Group (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Aprotinin, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer ripa buffer, by Merck Group (1 mentions)
Mp96 microplate reader spectrophotometer, by Safas (1 mentions)
Dx diagnostic, by BioLegend (1 mentions)
5 protocols using bradford colorimetric method
1
Protein Extraction from Microglial Cells
2
Protein Expression Analysis in Mice
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To examine protein expression, mice were killed, and tissues were removed after 7, 14, 21, and 28 days from surgery. Samples were homogenized in a lysis buffer containing 25 mM Tris-HCl pH (7.5), 25 mM NaCl, 5 mM EGTA, 2.5 mM EDTA, 2 mM NaPP, 4 mM PNFF, 1 mM di Na3VO4, 1 mM PMSF, 20 μg/mL leupeptin, 50 μg/mL aprotinin, and 0.1% SDS (Sigma-Aldrich). The homogenate was centrifuged at 12,000g for 30 minutes at 4°C, and the pellet was discarded. Proteins from cells were extracted by RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl 1% sodium deoxycholate, 1% Triton X-100, and 2 mM PMSF) (Sigma-Aldrich), and the insoluble pellet was separated by centrifugation (12,000g for 30 minutes, 4°C). The total protein concentration in the supernatant was measured using the Bradford colorimetric method (Sigma-Aldrich).41 (link)
3
Protein Extraction from Tissues and Cells
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Proteins extraction from tissues and cells was performed as previously reported [32 (link)]. Briefly, mice were sacrificed 7 days after SNI, and the lumbar spinal cord tissue was removed. Samples were homogenized in a lysis buffer containing 0.1% SDS (Sigma-Aldrich, St. Louis, MI, USA). The homogenate was centrifuged at 12,000× g for 30 min at 4 °C, and the pellet was discarded. Proteins from BV2 cells were extracted by radioimmunoprecipitation assay buffer (RIPA) buffer (Sigma-Aldrich), and the insoluble pellet was separated by centrifugation (12,000× g for 30 min, 4 °C). The total protein concentration in the supernatant was measured using the Bradford colorimetric method (Sigma-Aldrich, St. Louis, MO, USA).
4
Evaluating MAPK Activation in BV-2 Cells
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MAPKs (ERK1/2, JNK, and p38) activation was evaluated using non‐competitive sandwich ELISA (Biolegend e‐Bioscience DX Diagnostic, MA), according to the manufacturer's instructions. BV‐2 cells (1 × 105 cells/well) were seeded into 24‐well plates and cultured for 24 hr. Cells were pre‐treated for 4 hr with CSE (1 μg/ml) then stimulated with LPS (250 ng/ml) for 30, 60, and 120 min, and lysed with 100 μl of the provided lysis buffer. The total protein content of each sample was evaluated by Bradford colorimetric method (Merck KGaA; Germany), using bovine serum albumi (BSA) (Merck KGaA, Germany) as a reference standard. Absorbance was measured at 450 nm using an MP96 microplate reader spectrophotometer (Safas, Monaco). The activation of MAPKs was calculated as the ratio of phosphorylated to total proteins, normalizing values to the untreated control. Three independent experiments were performed.
5
Protein Expression Analysis in Animal Tissues
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In order to detect protein expression in the animals’ tissues, spinal cords were removed on day 14 post-surgery by separating the contralateral and ipsilateral sides. Samples were homogenized in a lysis buffer containing 25 mM Tris-HCl pH (7.5), 2.5 mM EDTA, 5 mM EGTA, 25 mM NaCl, 4 mM PNFF, 2 mM NaPP, 1 mM PMSF, 1 mM di Na3VO4, 50 μg/mL aprotinin, 20 μg/mL leupeptin, and 0.1% SDS (Merck, Darmstadt, Germany). The homogenate was centrifuged at 12,000× g for 30 min at 4 °C, and the total protein concentration was measured in the supernatant (Bradford colorimetric method; Merck, Darmstadt, Germany).
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