Three animals were sacrificed 24 h after ischemia by rapid decapitation under deep anesthesia. Brain tissues were prepared for western blotting. Protein concentration was determined by the Bradford assay (Tiangen Biotech Co., Ltd., Beijing, China). Protein samples (50 μg per lane) were electrophoresed in 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 60 V for 2 hours at 4°C in a transfer buffer containing 48 mmol L-1 Tris-base, 39 mmol L-1 glycine, and 20% methanol. The blots were blocked in fresh blocking buffer (Tris-buffered saline with 0.05% Tween 20 [TBS-T] plus 5% non-fat dry milk) for 1 h at room temperature. The blots were then incubated at 4°C overnight with anti-Atf2 antibody (sc-164978), anti-c-Fos antibody (sc-52), or anti-C/EBPγ antibody (sc-25769) (all at 1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin antibody (1: 10000, Santa Cruz Biotechnology). A secondary antibody conjugated with horseradish peroxidase (HRP, 1:5000, Bio-Rad) was used. Immunoblots were visualized on X-ray film by a chemiluminescence reaction (Pierce, Rockford, IL). Image analysis of the blots was performed on optical density-calibrated images using AlphaEase Stand Alone software (Alpha Innotech Corp., San Leandro, CA).
Bradford assay
Manufactured by Tiangen Biotech
Sourced in China
The Bradford assay is a laboratory technique used to measure the concentration of proteins in a sample. It is a colorimetric assay that relies on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a color change that can be measured spectrophotometrically. The Bradford assay provides a quick and efficient way to quantify protein levels in various biological samples.
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Lab products found in correlation
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp, by Bio-Rad (1 mentions)
Anti c fos antibody sc 52, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Glutathione (gsh), by Dojindo Laboratories (1 mentions)
Tbars assay kit, by Cayman Chemical (1 mentions)
E coli bl21 de3 strain, by Transgene (1 mentions)
Wright giemsa stain kit, by Nanjing Jiancheng (1 mentions)
5 protocols using bradford assay
1
Western Blotting for Brain Proteins
2
Quantifying Oxidative Stress Markers
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The intracellular MDA production was determined using a thiobarbituric acid reactive species (TBARS) assay kit (Cayman, USA). A total of 1 × 107 cells were homogenized in PBS, and the lysates were incubated with TBA color reagents at 50°C for 60 min. The MDA-TBA adducts were then measured colorimetrically at 540 nm. The total protein concentration was determined through the Bradford assay (Tiangen, China), and the results are expressed as [MDA] in nmol per mg of protein. GSH was determined according to the recycling system through the reaction of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and GSH (Dojindo, Japan). The concentration of GSH in the sample solutions was determined using a calibration curve and is expressed as nmol of GSH per mg of protein. The SOD activity was measured using an assay kit (Dojindo, Japan) according to the manufacturer's protocol. The CAT activity was assayed according to the method developed by Góth and is expressed as mU per mg of protein based on the rate of decrease of the hydrogen peroxide [21 (link)].
3
Protein Extraction and Quantification
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For each sample, 5×106 cells were harvested and lysed in 1 ml lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DL-Dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride (PMSF), 30 mM sodium fluoride, and 0.25 mg/ml RNaseA) for 1 h at room temperature, and then centrifuged at 4°C for 30 min. at 12,000 rpm. The supernatant was transferred to a new tube and concentrated with 0.1 M NH4COOH. Lysis buffer was added to a final volume of 800 µl, and the protein concentration was determined using a Bradford assay (Tiangen, Beijing, China).
4
E. coli Protein Expression and Purification
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E. coli BL21 (DE3) strain (TransGen Biotech, Beijing, China) was used for protein expression [58 (link)]. The plasmids of G6046 or its truncations were transformed into E. coli BL21 (DE3) strain, and the single clone was amplified to 1 L LB medium (100 μg/mL kanamycin or 100 μg/mL ampicillin). The cells were cultured at 37 °C and IPTG was added to a final concentration of 0.5 mM when OD600 = 0.8. Cells were incubated for another 20 h at 16 °C. The cells were harvested by centrifugation and stored at −40 °C for further use.
The recombinant G6046 protein was purified using the affinity chromatography method. The cells were re-suspended in the lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol) and then disrupted by sonication for 30 min (5 s on, 10 s off) on ice. The cell lysate was centrifuged at 16,000× g for 1 h. Two milliliters of Ni-NTA resin was added to a gravity column and equilibrated with the lysis buffer. The supernatant was loaded onto the column and washed with 50 mL of the lysis buffer containing 20 mM imidazole. The protein was eluted with 20 mL of the lysis buffer supplemented with 200 mM imidazole. The concentration of eluted G6046 protein was measured using the Bradford assay (TIANGEN biotech Beijing Co., Ltd., Beijing, China) and the purity was evaluated by SDS-PAGE.
The recombinant G6046 protein was purified using the affinity chromatography method. The cells were re-suspended in the lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol) and then disrupted by sonication for 30 min (5 s on, 10 s off) on ice. The cell lysate was centrifuged at 16,000× g for 1 h. Two milliliters of Ni-NTA resin was added to a gravity column and equilibrated with the lysis buffer. The supernatant was loaded onto the column and washed with 50 mL of the lysis buffer containing 20 mM imidazole. The protein was eluted with 20 mL of the lysis buffer supplemented with 200 mM imidazole. The concentration of eluted G6046 protein was measured using the Bradford assay (TIANGEN biotech Beijing Co., Ltd., Beijing, China) and the purity was evaluated by SDS-PAGE.
5
Bronchoalveolar Lavage Fluid Analysis
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BALF was performed immediately following euthanasia of the mice by cervical dislocation. After the trachea was exposed, the lungs were lavaged four times with 0.8 ml of cold sterile PBS, and the BALF was centrifuged at 1500 g for 10 min at 4°C. Supernatants were collected, and the total BALF protein was determined by standard Bradford assay (TIANGEN, China) according to the manufacturer's instruction. All leukocytes in the BALF were counted, 200 μl of which was cytospun onto slides, and the cells were stained with the Wright-Giemsa Stain Kit (Nanjing Jiancheng Bioengineering, China) for differential leukocyte identification under a microscope.
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