ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] utilized for measuring enzyme activity was purchased from Sigma-Aldrich. Bradford's reagent used for protein estimation was obtained from Fermentas, USA. The membrane separation unit (Ray-flow X100; Orelis, France) was used for enzyme filtration during reactor operations. Ultra-filtration membrane (10 kDa) was purchased from Permionics, India.
Bradford s reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bradford's reagent is a laboratory reagent used for the colorimetric determination of protein concentration. It is a dye-binding assay that measures the absorbance of the dye-protein complex at a specific wavelength, allowing for the quantification of protein levels in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent substrate, by Merck Group (2 mentions)
T per, by Thermo Fisher Scientific (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ls55 luminescence spectrophotometer, by PerkinElmer (1 mentions)
Total carbohydrate assay kit, by Merck Group (1 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (1 mentions)
Albumin, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Phenol, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
X omat film, by Kodak (1 mentions)
9 protocols using bradford s reagent
1
Enzyme Activity Measurement Protocol
2
Transcriptional Changes in Rat Brain Regions
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Translational expression changes in frontal cortex and hippocampus region of rat brain following the exposure of RV were assayed following the method described earlier by us63 (link). The brain tissue was homogenized in RIPA buffer and further processed for western blot analysis. After protein estimation by Bradford’s Reagent (Fermentas Inc., Glen Burnie, MD), equal amounts (40 μg/well) of denatured proteins were loaded onto Tricine–SDS/SDS-PAGE gel and the rest of the procedure used was identical as described in the in vitro section. Expression levels SIRT1, p-ERK, ERK1/2 p-p38, p38 and p-JNK, p-TrkA, TrkA, p75NTR, activated caspase-3 (1:1000, Chemicon) and β-actin (1:2000, Sigma) were probed in these tissue samples.
3
Microsome Preparation and Enzyme Activity Assay
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Following the respective exposures cells were harvested and processed for microsome preparation following the protocol described earlier by us [13] (link). In brief, cells were scrapped in PBS at 4°C and pelleted by centrifuging at 500×g for 10 min. The cell pellet was resuspended in microsomal dilution buffer containing 0.1% (v/v) glycerol, 0.25 mM protease inhibitors cocktail, 0.01M EDTA and 0.1 mM dithiothreitol. The cells were then sonicated thrice at 15 Hz for 10 seconds each. Following sonication, the cells were again centrifuged at 9000×g for 20 min. The supernatant was then further centrifuged at 105,000×g for 60 min, to isolate the microsomal fraction. The microsomal pellet, thus obtained was then resuspended in microsomal dilution buffer and protein estimation was done by Bradford's Reagent (Fermentas Inc., Maryland, USA). The activity of 7-ethoxyresorufin-O-deethylase (EROD) for CYP1A1, 7-pentoxyresourfin-O-dealkylase (PROD) for CYP2B6, and N-nitrosodimethylamine demethylase (NDMA-d) for CYP2E1 were determined by following the methods described earlier by us [12] (link), [13] (link), [14] (link), [52] (link) using a Perkin Elmer LS 55 Luminescence spectrophotometer.
4
Protein and Carbohydrate Quantification in Jatropha Transgenics
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Protein content of Jatropha transgenic lines was determined as described by Focks and Benning [69 (link)] using 200 mg of each leaf and dried endosperm. Protein amounts were measured according to Bradford [70 (link)] using three technical replications using ready to use Bradford’s reagent (Fermentas, USA). To analyze the carbohydrate content, 200 mg of each of leaf and dried endosperm were homogenized in 200 μL of assay buffer and centrifuged at full speed. The extracted supernatant was used for quantification of carbohydrate using a Total Carbohydrate Assay Kit (Sigma-Aldrich). d -glucose was used as a standard for calibration, and data were expressed as mg carbohydrate g−1 tissue fresh weight.
5
Protein Extraction for ELISA and RBD
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Fresh callus (150 mg) was ground under liquid nitrogen in three volumes (v/w) of phosphate-buffered saline (PBS, pH 7.4) for ELISA and Western blot analysis, or in five volumes (v/w) of PBS with 20 mM imidazole (pH 7.4) for RBD purification. Samples were vortexed and centrifuged twice (10,000× g, 10 min, 4 °C). The supernatant was collected and the total protein concentration was determined using Bradford’s reagent (Thermo Fisher Scientific, Waltham, MA, USA).
6
Gelatin Zymography Protocol
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Cell-free conditioned medium was collected for zymography, after 36 h of exposure. Protein concentration of the conditioned medium was estimated using Bradford’s reagent (Thermoscientific). Thirty five microgram protein was loaded and resolved in 10% polyacrylamide gel containing 1.5% gelatin substrate. The gels were washed in 2.5% Triton-X 100 for 30 min and in Milli-Q water thrice at an interval of 10 min, followed by incubation in developing buffer (50 mM Tris-HCL pH 7.5 containing 200 mM NaCl, 10 mM CaCl2, 0.05% Brij-35 and 0.02% Sodium azide) for 18 h at 37°C. Gels were stained using Coomassie brilliant blue-R 250 solution containing 40% methanol and 10% acetic acid. A mixture of 40% methanol and 10% acetic acid without Coomassie-R 250 was used for destaining the gel. The gelatinolytic activity was visualized as a zone of clearance against a blue background. Gels were scanned using GS800-calibrated densitometer (BioRad, Hercules, CA USA).
7
Milk Kefir Grains Production Analysis
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The milk kefir grains and plastic sieves were from Kefiralia (Arrasate, Gipuzkoa, Spain). They were composed, according to the label provided by the manufacturer, of 109 CFU/g of LAB (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar diacetylactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Lactobacillus kefyr), Candida kefyr, and Saccharomyces unisporus subsp. The milks used were purchased with the following brands: Granarolo (UHT [ultra-high temperature] cow milk), Eurospin (UHT goat milk) and fresh buffalo milk (Naples, Italy) that had been pasteurized at 90 °C for 15 min before use.
Albumin (analytical standard) and Bradford’s reagent were purchased from Thermo Fisher (Milan, Italy); sulfuric acid, phenol, buffer solutions for pH meter calibration (pH 4 and 7), and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Milan, Italy).
Albumin (analytical standard) and Bradford’s reagent were purchased from Thermo Fisher (Milan, Italy); sulfuric acid, phenol, buffer solutions for pH meter calibration (pH 4 and 7), and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Milan, Italy).
8
Western Blot Analysis of Liver Proteins
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Liver tissue was directly lysed with an extraction buffer (T-PER, Thermo Fisher Scientific Inc., Rockford, lL, USA) for 30 min on ice. After centrifugation at 13,000 g for 15min at 4°C, protein concentration in the supernatant was measured using Bradford’s reagent (Thermo Fisher Scientific Inc.). Protein (30 μg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a gradient gel and then transferred onto PVDF membranes. Blocking was carried out using blocking buffer [5% nonfat dairy milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4) containing 0.05% Tween-20] for 1 hr at room temperature. Primary antibodies were diluted 1:1,000 in a blocking buffer and incubated at 4°C overnight. The following antibodies were used: anti-LTA (mouse antibody, Invitrogen, Waltham, MA, USA). To detect antigen antibody complexes, anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, TX, USA) were diluted 1:3000 in blocking buffer and incubated at room temperature for 45 min. Immune complexes were visualized using chemiluminescent substrate (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions.
9
Protein Expression Analysis in Liver Tissue
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Liver tissue was directly lyzed with an extraction buffer (T-PER, Thermo Fisher Scientific Inc.) for 30 minutes on ice. After centrifugation at 13,000 Â g for 15 minutes at 4 C, protein concentration in the supernatant was measured using Bradford's reagent (Thermo Fisher Scientific Inc.). Protein (30 mg) was resolved by sodium dodecyl sulfateepolyacrylamide gel electrophoresis on a gradient gel and then transferred onto polyvinylidene difluoride membranes. Blocking was performed using blocking buffer [5% nonfat dairy milk in Tris-buffered saline (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.4) containing 0.05% Tween -20] for 1 hour at room temperature. Primary antibodies were diluted 1:1000 in a blocking buffer and incubated at 4 C overnight. The following antibodies were used: anti-Bcl-2, anti-Bax, and b-actin (mouse or rabbit antibody, Cell Signaling Technology, Danvers, MA). To detect antigen antibody complexes, anti-rabbit or anti-mouse horseradish peroxidaseeconjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, TX) were diluted 1:3000 in blocking buffer and incubated at room temperature for 45 minutes. Immune complexes were visualized using chemiluminescent substrate (Millipore, Burlington, MA) and Kodak X-OMAT film (Eastman Kodak, Rochester, NY) according to the manufacturer's instructions.
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