Cortical cytosolic protein extraction was conducted using a protocol adapted from Vasconcelos et al.51 (link). Briefly, tissues were homogenized in a glass-glass Dounce homogenizer in ice-cold lysis buffer (20 mM HEPES, 1.0 mM MgCl2, 0.5 mM EDTA, 1% NP-40, 1.0 mM EGTA, 0.5 mM PMSF, 2 g/mL leupeptin, 2 g/mL antipain, 3 mM Na3VO4, 20 mM sodium pyrophosphate) and centrifuged at 17,000 × g for 5 min at 4 °C. The supernatant was collected and stored at −80 °C for western blot analyses. Protein concentration was determined using the Bradford colorimetric method (#500-0006, Bio-Rad, Hercules, CA, USA)52 (link).
Bradford colorimetric method
Manufactured by Bio-Rad
Sourced in United States
The Bradford colorimetric method is a laboratory technique used for the quantitative determination of protein concentration in a sample. It is a simple, rapid, and sensitive assay that relies on the binding of the Coomassie Brilliant Blue dye to proteins, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Speed vacuum system, by Martin Christ (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride pvdf membrane, by GE Healthcare (1 mentions)
Anti ugpase polyclonal antibody, by Agrisera (1 mentions)
Complete edta free, by Roche (1 mentions)
Quantiglo, by R&D Systems (1 mentions)
Mcp 1 elisa kits, by BD (1 mentions)
Icam 1, by R&D Systems (1 mentions)
8 protocols using bradford colorimetric method
1
Cortical Cytosolic Protein Extraction
2
Protein Extraction and Quantification
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Total protein was extracted from the colonies as described previously [20 (link)]; protein concentration was determined with Bradford colorimetric method (Bio-Rad) according to standard protocol. At least 100 μg of protein from each sample was lyophilized using a speed vacuum system (Martin Christ, Germany).
3
Protein Extraction and Western Blot Analysis
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Total protein extracts were prepared from 3-day-old etiolated seedlings maintained in darkness or irradiated with 20 μmol m−2 s−1 of blue light for 15 min. Under a dim red safe light, plant tissue was ground in a mortar and pestle in extraction buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor mixture (Complete EDTA-free; Roche) and clarified by centrifugation at 10,000 g, 4°C for 10 min. The resulting supernatant was used as the total protein extract. Protein concentrations were determined by the Bradford colorimetric method (Bio-Rad). All samples were mixed with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.004% bromophenol blue), boiled for 4 min and subjected to 7.5% SDS-PAGE. Proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (GE Healthcare) by electroblotting and detected with anti-phot1 polyclonal antibody (Cho et al., 2007 (link)), anti-NPH3 polyclonal antibody (Tsuchida-Mayama et al., 2008 (link)), and anti-UGPase polyclonal antibody (Agrisera). Blots were developed with horseradish peroxidase (HRP)-linked secondary antibodies (Promega) and Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).
4
Measuring Biomarkers in Plasma and Urine
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Creatinine concentration was measured in plasma and urine by the picric acid method adapted for microtiter plates (Allcock et al. 1998 ). Urinary ET‐1 concentration was measured by chemiluminescent immunoassay (QuantiGlo, R&D Systems, Minneapolis, MN). Thiobarbituric acid reactive substances (TBARS) in plasma and urine were quantified using an OXItek assay kit (ZeptoMetrix, Buffalo, NY). Urinary protein concentration was determined using the Bradford colorimetric method (Bio‐Rad Laboratories, Hercules, CA).
5
Quantification of VEGF and MCP-1 in Retinal Lysates
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VEGF and MCP-1 were measured as described previously [37 (link)]. Briefly, supernatants from retinal lysates were collected, and the total protein concentration was quantitated using the Bradford colorimetric method (Bio-Rad Laboratories, NSW, Australia) according to the manufacturer’s protocol. Undiluted supernatants were then assayed using a commercially available rat VEGF, ICAM-1 (R&D Systems, MN, USA), and MCP-1 ELISA kits (BD Biosciences, CA, USA). All procedures were performed according to the manufacturer’s instructions. Six rats per group were evaluated.
6
Extraction and Quantification of Active RhoA
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To obtain protein lysates, the cells were plated on 10 mm dishes at approximately 60% confluence. Following radiation treatment for the specified durations and at the specified doses, the cells were washed twice with ice-cold PBS and disrupted with RIPA lysis buffer (50 mM Tris, pH 7.2; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 500 mM NaCl; 10 mM MgCl2; 1 mM Na3Vo4; 1 mM NaF; 1 mM PMSF; and 10 μg/mL each of aprotinin and leupeptin) and stored at −20°C. The protein concentration was quantified using the Bradford colorimetric method (Bio-Rad). A 500 μg sample of the total lysate was subsequently incubated in 25 μg of RBD-GST at 4°C for 90 min. Then, the beads were centrifuged at 4°C for 3 min, washed three times with buffer B (Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 1 mM Na3Vo4, 1 mM NaF, 1 mM PMSF, and 10 μg/mL each of aprotinin and leupeptin), and intercalated via centrifugation at 3,000 rpm for 3 min in a cold room. The active RhoA protein (RhoA-GTP) bound to the glutathione-Sepharose beads was detected via Western blotting [26 (link)].
7
Quantifying Melatonin Levels in Bone Marrow and Spleen
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The content of melatonin in BM was obtained from the femur by flushing with 1 ml of cold PBS, the fluid was centrifuged (14000 g, 5 min at 4 °C) and supernatant was collected. For the spleen melatonin, half spleen was homogenized in 2 mM Tris-HCl buffer (adding 1 mM EDTA and 1 mM EGTA) in a ratio of 130 mg of tissue to 200 μl of Tris-HCl buffer; the homogenate was then centrifuged (14000 g, 5 min at 4 °C) and the supernatant was collected.
Melatonin was measured using ELISA kits following the manufacturer’s instructions (Immuno Biological Laboratories, Hamburg, Germany). The detection limits of the melatonin ELISA kits were 0.5 pg/ml. Values were relativized to the amount of protein, as measured by the Bradford colorimetric method (BioRad, Hercules, CA, USA).
Melatonin was measured using ELISA kits following the manufacturer’s instructions (Immuno Biological Laboratories, Hamburg, Germany). The detection limits of the melatonin ELISA kits were 0.5 pg/ml. Values were relativized to the amount of protein, as measured by the Bradford colorimetric method (BioRad, Hercules, CA, USA).
8
Cortical tissue homogenization and protein extraction
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Cortical tissues were homogenized in a glass-glass Dounce homogenizer in ice-cold lysis buffer (20 mM HEPES, 1.0 mM MgCl2, 0.5 mM EDTA, 1% NP-40, 1.0 mM EGTA, 0.5 mM PMSF, 2 g/mL leupeptin, 2 g/mL antipain, 3 mM Na3VO4, and 20 mM sodium pyrophosphate) and centrifuged at 17,000×g for 5 min at 4 °C. The supernatant was collected and stored at − 80 °C for western blot analysis. The protein concentration was determined using the Bradford colorimetric method (#500-0006, Bio-Rad, Hercules, CA, USA).
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