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Dinitrophenylhydrazine
Dinitrophenylhydrazine
Dinitrophenylhydrazine is a chemical reagent used in organic synthesis and analytical chemistry.
It reacts with carbonyl compounds to form colored hydrazone derivatives, enabling the identification and quantification of these compounds.
Researchers can leverage PubCompare.ai's AI-powered platform to streamline their workflow when working with dinitrophenylhydrazine, locating protocols from literature, preprints, and patents, and using AI-driven comparisons to identify the best methodologies and produts.
This innovative tool enhances reproducibility and research accuracy for studies involving dinitrophenylhydrazine.
It reacts with carbonyl compounds to form colored hydrazone derivatives, enabling the identification and quantification of these compounds.
Researchers can leverage PubCompare.ai's AI-powered platform to streamline their workflow when working with dinitrophenylhydrazine, locating protocols from literature, preprints, and patents, and using AI-driven comparisons to identify the best methodologies and produts.
This innovative tool enhances reproducibility and research accuracy for studies involving dinitrophenylhydrazine.
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sodium borohydride
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The static ORP (sORP) marker was determined using the RedoxSYS diagnostic system (Luoxis Diagnostics, Inc., Englewood, CO, USA) as previously described (17 (link),18 (link)). This value is indicative of the integrated balance of oxidants and reductants in a specimen and is presented in mV. Using this innovative method, 20 µl of plasma were applied to disposable sensors designed by Luoxis Diagnostics, Inc., which were inserted into the RedoxSYS diagnostic system and the sORP value was reported within 4 min.
For the determination of the levels of TBARS, an assay was used based on the study by Keles et al (19 (link)). TBARS is a commonly and frequently used method to determine the lipid peroxidation (20 (link)). In accordance with this method, 100 µl of plasma were mixed with 500 µl of 35% trichloroacetic acid (Merck KGaA, Darmstadt, Germany) and 500 µl of Tris-HCl (Sigma-Aldrich, St. Louis, MO, USA; 200 mmol/l, pH 7.4) followed by incubation for 10 min at room temperature. A total of 1 ml of 2 M sodium sulfate and 55 mmol/l TBA solution were added and the samples were then incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed following the addition of 1 ml of 70% TCA. The samples were centrifuged at 15,000 × g for 3 min and the absorbance of the supernatant was read at 530 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi, Tokyo, Japan). A baseline absorbance was taken into account by running a blank along with all samples during the measurement. The calculation of the TBARS concentration was based on the molar extinction co-efficient of malondialdehyde.
The GSH concentration was measured as previously described in the study by Reddy et al (21 ). A total of 20 µl of erythrocyte lysate treated with 5% TCA was mixed with 660 µl of 67 mmol/l sodium potassium phosphate (pH 8.0) and 330 µl of 1 mmol/l 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The samples were then incubated in the dark at room temperature for 45 min and the absorbance was read at 412 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The GSH concentration was calculated on the basis of calibration curves made using commercial standards.
The concentration of CARB, an index of protein oxidation, was determined based on the method described in the study by Patsoukis et al (22 (link)). In this assay, 50 µl of 20% TCA were added to 50 µl of plasma and this mixture was then incubated in an ice bath for 15 min and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 500 µl of 10 mmol/l 2,4-dinitrophenylhydrazine (DNPH; in 2.5 N HCl) for the sample, or 500 µl of 2.5 N HCl for the blank, were added to the pellet. The samples were incubated in the dark at room temperature for 1 h with intermittent vortexing every 15 min and were centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of 10% TCA was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of ethanol-ethyl acetate (1:1 v/v) was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. This washing step was repeated twice. The supernatant was discarded and 1 ml of 5 M urea (pH 2.3) was added, vortexed and incubated at 37°C for 15 min. The samples were centrifuged at 15,000 × g for 3 min at 4°C and the absorbance was read at 375 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The calculation of the CARB concentration was based on the molar extinction co-efficient of DNPH. Total plasma protein was assayed using Bradford reagent (Sigma, Hamburg, Germany).
The determination of TAC was based on the method described in the study by Janaszewska and Bartosz (23 (link)). Briefly, 20 µl of plasma were added respectively to 480 µl of 10 mmol/l sodium potassium phosphate (pH 7.4) and 500 µl of 0.1 mmol/l 1,1-diphenyl-1-picrylhydrazyl (DPPH) and the samples were incubated in the dark for 60 min at room temperature. The samples were then centrifuged for 3 min at 20,000 × g and the absorbance was read at 520 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi).
The measurement of CAT activity was carried out as previously described by Aebi (24 (link)). In particular, 4 µl οf erythrocyte lysate (diluted 1:10) were added to 2,991 µl οf 67 mmol/l sodium potassium phosphate (pH 7.4) and the samples were incubated at 37°C for 10 min. A total of 5 µl of 30% H2O2 was added to the samples and the change in absorbance was immediately read at 240 nm [using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi)] for 130 sec. The calculation of CAT activity was based on the molar extinction co-efficient of H2O2. Each assay was performed twice in triplicate.
For the determination of the levels of TBARS, an assay was used based on the study by Keles et al (19 (link)). TBARS is a commonly and frequently used method to determine the lipid peroxidation (20 (link)). In accordance with this method, 100 µl of plasma were mixed with 500 µl of 35% trichloroacetic acid (Merck KGaA, Darmstadt, Germany) and 500 µl of Tris-HCl (Sigma-Aldrich, St. Louis, MO, USA; 200 mmol/l, pH 7.4) followed by incubation for 10 min at room temperature. A total of 1 ml of 2 M sodium sulfate and 55 mmol/l TBA solution were added and the samples were then incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed following the addition of 1 ml of 70% TCA. The samples were centrifuged at 15,000 × g for 3 min and the absorbance of the supernatant was read at 530 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi, Tokyo, Japan). A baseline absorbance was taken into account by running a blank along with all samples during the measurement. The calculation of the TBARS concentration was based on the molar extinction co-efficient of malondialdehyde.
The GSH concentration was measured as previously described in the study by Reddy et al (21 ). A total of 20 µl of erythrocyte lysate treated with 5% TCA was mixed with 660 µl of 67 mmol/l sodium potassium phosphate (pH 8.0) and 330 µl of 1 mmol/l 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The samples were then incubated in the dark at room temperature for 45 min and the absorbance was read at 412 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The GSH concentration was calculated on the basis of calibration curves made using commercial standards.
The concentration of CARB, an index of protein oxidation, was determined based on the method described in the study by Patsoukis et al (22 (link)). In this assay, 50 µl of 20% TCA were added to 50 µl of plasma and this mixture was then incubated in an ice bath for 15 min and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 500 µl of 10 mmol/l 2,4-dinitrophenylhydrazine (DNPH; in 2.5 N HCl) for the sample, or 500 µl of 2.5 N HCl for the blank, were added to the pellet. The samples were incubated in the dark at room temperature for 1 h with intermittent vortexing every 15 min and were centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of 10% TCA was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of ethanol-ethyl acetate (1:1 v/v) was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. This washing step was repeated twice. The supernatant was discarded and 1 ml of 5 M urea (pH 2.3) was added, vortexed and incubated at 37°C for 15 min. The samples were centrifuged at 15,000 × g for 3 min at 4°C and the absorbance was read at 375 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The calculation of the CARB concentration was based on the molar extinction co-efficient of DNPH. Total plasma protein was assayed using Bradford reagent (Sigma, Hamburg, Germany).
The determination of TAC was based on the method described in the study by Janaszewska and Bartosz (23 (link)). Briefly, 20 µl of plasma were added respectively to 480 µl of 10 mmol/l sodium potassium phosphate (pH 7.4) and 500 µl of 0.1 mmol/l 1,1-diphenyl-1-picrylhydrazyl (DPPH) and the samples were incubated in the dark for 60 min at room temperature. The samples were then centrifuged for 3 min at 20,000 × g and the absorbance was read at 520 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi).
The measurement of CAT activity was carried out as previously described by Aebi (24 (link)). In particular, 4 µl οf erythrocyte lysate (diluted 1:10) were added to 2,991 µl οf 67 mmol/l sodium potassium phosphate (pH 7.4) and the samples were incubated at 37°C for 10 min. A total of 5 µl of 30% H2O2 was added to the samples and the change in absorbance was immediately read at 240 nm [using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi)] for 130 sec. The calculation of CAT activity was based on the molar extinction co-efficient of H2O2. Each assay was performed twice in triplicate.
For protein extraction mid-jejunum mucosa samples were thawed on ice and 1 g of the sample was placed in sterile tube (5 mL tube, Eppendorf, Hamburg, Germany) followed by addition of 2 mL of PBS (MP Biomedicals, Inc., Santa Ana, CA, USA). Samples were homogenized (Tissuemiser, Thermo Fisher Scientific, Waltham, MA, USA) for 30 s and centrifuged at 87,000× g for 20 min on ice. The supernatant was subdivided into vials stored at −80 °C until being used to evaluate antioxidant status, immune response, and intestinal barrier function in mid-jejunum mucosa relative to the protein content of samples (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). Protein quantification started by mixing 25 µL of each sample with 200 µL of working reagent provided in the kit in a microplate well (96-Well EIA/RIA Plates, Corning, Corning, NY, USA), followed by 30 s incubation in plate shaker. The plate was covered with clear adhesive strip and incubated for 30 min at 37 °C. The plate was cooled to room temperature and wells were read at 562 nm.
The quantification of protein carbonyls (STA-310, Cell Biolabs, Inc., San Diego, CA, USA) as an index of oxidized proteins is described by Shen et al. [89 (link)]. Briefly, the protein content of each sample determined in the previous assay was diluted to 10 µg/mL. Diluted samples (100 µL) were pipetted into wells and incubated for 2 h at 37 °C. Each well was washed three times with 250 µL of PBS (MP Biomedicals, Inc., Santa Ana, CA, USA) and 100 µL of working solution supplied in the kit added before plate incubation in the dark for 45 min. Each well was washed with 250 µL of PBS/ethanol (1:1, v/v) and incubated for 5 min in an orbital shaker; this procedure was repeated four times. Each well was washed with 250 µL of PBS twice, 200 µL of blocking solution was added, and the plate was incubated for 1 h in an orbital shaker. Each well was washed with 250 µL of washing buffer three times and 100 µL of anti-dinitrophenylhydrazine antibody supplied in the kit were added according to dilutions recommended by the manufacturer. The plate was incubated in an orbital shaker for 1 h. Each well was washed with 250 µL of washing buffer three times and 100 µL of horseradish peroxidase antibody were added for incubation for 1 h in an orbital shaker. Each well was washed with 250 µL of washing buffer five times, 100 µL of substrate were added, and 100 µL of stop solution were added after the onset color development. The wells were read at 450 nm.
Malondialdehydes (STA-330, Cell Biolabs, Inc., San Diego, CA, USA) were measured by incubating for 5 min 100 µL of each sample in equal volume of SDS lysis solution provided in the kit. Followed by incubation at 95 °C for 45 min with 250 µL of the reagent (130 mg of thiobarbituric acid in 25 mL of diluent) supplied in the kit, which had the pH adjusted (Accumet AB15 pH Meter, Fisher Scientific, Hampton, NH, USA) to 3.5 with sodium hydroxide. Tubes were cooled in for 5 min and centrifuged at 4000× g for 15 min. The supernatant (300 µL) was vigorously mixed with 300 µL of butanol for 2 min and centrifuged at 10,000× g for 5 min. The supernatant (200 µL) was transferred to a microplate (96-Well EIA/RIA Plates, Corning, Corning, NY, USA) and samples were read at 532 nm.
Tumor necrosis factor-α (PTA00, R&D Systems, Inc., Minneapolis, MN, USA) was measured by pipetting 50 µL of assay diluent supplied in the kit with 50 µL of samples into wells. The plate was covered with clear adhesive strip and incubated for 2 h. Each well was washed five times with 300 µL of washing buffer, 100 µL of TNF-α conjugate supplied in the kit were added, and the plate was incubated following same specifications. Each well was washed five times with 300 µL of washing buffer, 100 µL of substrate solution supplied in the kit were added to each well, and the plate was incubated for 30 min in the dark. After incubation, 100 µL of stop solution supplied in the kit were added and wells were read 450 and 570 nm to obtain reading at 570 subtracted from 450 nm.
Iterleukin-8 quantification (P8000, R&D Systems, Inc., Minneapolis, MN, USA) was performed by pipetting 50 µL of assay diluent supplied in the kit with 100 µL of samples into wells. The plate was covered with clear adhesive strip and incubated for 2 h in orbital shaker at 500 rpm. Each well was washed five times with 300 µL of washing buffer, 200 µL of porcine IL-8 conjugate supplied in the kit were added, and the plate was incubated following same specifications. Each well was washed five times with 300 µL of washing buffer, 120 µL of substrate solution supplied in the kit were added, and the plate incubated for 30 min in the dark. After incubation, 120 µL of stop solution supplied in the kit were added and wells were read 450 and 570 nm to obtain reading at 570 subtracted from 450 nm.
Immunoglobulin A (E100-102, Bethyl Laboratories, Inc., Montgomery, TX, USA) and IgG (E100-104, Bethyl Laboratories, Inc., Montgomery, TX, USA) were measured by pipetting 100 µL of their respective affinity purified antibody in each well according to the kit dilution. The plate was incubated for 1 h. Each well was washed five times with 260 µL of washing buffer supplied in the kit, 200 µL of blocking buffer supplied in the kit were added, and the plate was incubated for 30 min. Each well was washed five times with 260 µL of washing buffer, 100 µL of samples were added and incubated for 30 min. Each well was washed five times with 260 µL of washing buffer, 100 µL of diluted horseradish peroxidase supplied in the kit were added, and the plate was incubated for 1 h. Each well was washed five times with 260 µL of washing buffer, 100 µL of tetramethylbenzidine substrate were added, and the plate was incubated in the dark for 15 min. Sulfuric acid (100 µL) at 0.18 M was used as stop solution. The plate was read at 450 nm.
For measurement of total glutathione, a different protein extraction method was used, as recommended by the kit manufacturer total glutathione (STA-312, Cell Biolabs, Inc., San Diego, CA, USA). Mid-jejunum mucosa (100 mg) and 1 mL of metaphosphoric acid at 5% were mixed and homogenized with a glass pestle. The homogenate was centrifuged at 64,000× g for 15 min. The supernatant was used for total glutathione determination total glutathione (STA-312, Cell Biolabs, Inc., San Diego, CA, USA). Glutathione reductase solution (25 µL), NADPH solution (25 µL) supplied in the kit, and samples (100 µL) were added to each well. The chromogen solution (100 µL) supplied in the kit was added to each well and the plate was read at 405 nm every 2 min during 10 min. All wavelengths (for quantifications of protein, protein carbonyls, malondialdehydes, total glutathione, TNF-α, IL-8, IgA, and IgG) were read at the same microplate reader (Synergy HT, Biotek, Winooski, VT, USA).
Ileal digesta was freeze dried (SP Scientific, Virtis 24DX48 GPFD/300820, Warminster, PA, USA) and ground. Subsamples of ground material were analyzed for apparent ileal digestibility of dry matter [90 (link)], gross energy (6200 Calorimeter, Parr Instrument Company, Moline, IL, USA), nitrogen (method 990.03, [91 ], ATC Scientific, North Little Rock, AR, USA), and ether extract (method 920.39, [91 ]).
Fixed mid-jejunal tissue was removed from 10% buffered formaldehyde after two weeks for the obtainment of two transversal cuts that were transferred histological cassettes and submerged in 70% ethanol. Mid-jejunal cuts were included in paraffin for assembling histological slides after staining for Ki-67 antigen. The immunohistochemistry staining with Ki-67 primary monoclonal antibody (1:500 dilution) followed by anti-mouse secondary antibody (1:2 dilution factor) and the use of diamino-benzamine reagent for color development was performed in accordance with methods previously described by Kim et. al. [20 (link)]. Ten pictures of each pig were used to measure gut morphology by a single researcher choosing a well-oriented villus and its associated crypt. Measurements included: villus width (at half of villus height), villus height (from tip of the villus to top of the crypt), crypt depth (from top to bottom of the crypt), and calculating villus height: crypt depth [86 (link)]. The proportion of proliferating cells in the crypt was also estimated by calculating the proportion of cells positive to Ki-67 after taking pictures at 40× in Sony Van–Ox S microscope (Opelco, Washington, DC, USA) and processing in ImageJS tool [92 (link)] for analysis as described by Holanda and Kim [86 (link)].
The quantification of protein carbonyls (STA-310, Cell Biolabs, Inc., San Diego, CA, USA) as an index of oxidized proteins is described by Shen et al. [89 (link)]. Briefly, the protein content of each sample determined in the previous assay was diluted to 10 µg/mL. Diluted samples (100 µL) were pipetted into wells and incubated for 2 h at 37 °C. Each well was washed three times with 250 µL of PBS (MP Biomedicals, Inc., Santa Ana, CA, USA) and 100 µL of working solution supplied in the kit added before plate incubation in the dark for 45 min. Each well was washed with 250 µL of PBS/ethanol (1:1, v/v) and incubated for 5 min in an orbital shaker; this procedure was repeated four times. Each well was washed with 250 µL of PBS twice, 200 µL of blocking solution was added, and the plate was incubated for 1 h in an orbital shaker. Each well was washed with 250 µL of washing buffer three times and 100 µL of anti-dinitrophenylhydrazine antibody supplied in the kit were added according to dilutions recommended by the manufacturer. The plate was incubated in an orbital shaker for 1 h. Each well was washed with 250 µL of washing buffer three times and 100 µL of horseradish peroxidase antibody were added for incubation for 1 h in an orbital shaker. Each well was washed with 250 µL of washing buffer five times, 100 µL of substrate were added, and 100 µL of stop solution were added after the onset color development. The wells were read at 450 nm.
Malondialdehydes (STA-330, Cell Biolabs, Inc., San Diego, CA, USA) were measured by incubating for 5 min 100 µL of each sample in equal volume of SDS lysis solution provided in the kit. Followed by incubation at 95 °C for 45 min with 250 µL of the reagent (130 mg of thiobarbituric acid in 25 mL of diluent) supplied in the kit, which had the pH adjusted (Accumet AB15 pH Meter, Fisher Scientific, Hampton, NH, USA) to 3.5 with sodium hydroxide. Tubes were cooled in for 5 min and centrifuged at 4000× g for 15 min. The supernatant (300 µL) was vigorously mixed with 300 µL of butanol for 2 min and centrifuged at 10,000× g for 5 min. The supernatant (200 µL) was transferred to a microplate (96-Well EIA/RIA Plates, Corning, Corning, NY, USA) and samples were read at 532 nm.
Tumor necrosis factor-α (PTA00, R&D Systems, Inc., Minneapolis, MN, USA) was measured by pipetting 50 µL of assay diluent supplied in the kit with 50 µL of samples into wells. The plate was covered with clear adhesive strip and incubated for 2 h. Each well was washed five times with 300 µL of washing buffer, 100 µL of TNF-α conjugate supplied in the kit were added, and the plate was incubated following same specifications. Each well was washed five times with 300 µL of washing buffer, 100 µL of substrate solution supplied in the kit were added to each well, and the plate was incubated for 30 min in the dark. After incubation, 100 µL of stop solution supplied in the kit were added and wells were read 450 and 570 nm to obtain reading at 570 subtracted from 450 nm.
Iterleukin-8 quantification (P8000, R&D Systems, Inc., Minneapolis, MN, USA) was performed by pipetting 50 µL of assay diluent supplied in the kit with 100 µL of samples into wells. The plate was covered with clear adhesive strip and incubated for 2 h in orbital shaker at 500 rpm. Each well was washed five times with 300 µL of washing buffer, 200 µL of porcine IL-8 conjugate supplied in the kit were added, and the plate was incubated following same specifications. Each well was washed five times with 300 µL of washing buffer, 120 µL of substrate solution supplied in the kit were added, and the plate incubated for 30 min in the dark. After incubation, 120 µL of stop solution supplied in the kit were added and wells were read 450 and 570 nm to obtain reading at 570 subtracted from 450 nm.
Immunoglobulin A (E100-102, Bethyl Laboratories, Inc., Montgomery, TX, USA) and IgG (E100-104, Bethyl Laboratories, Inc., Montgomery, TX, USA) were measured by pipetting 100 µL of their respective affinity purified antibody in each well according to the kit dilution. The plate was incubated for 1 h. Each well was washed five times with 260 µL of washing buffer supplied in the kit, 200 µL of blocking buffer supplied in the kit were added, and the plate was incubated for 30 min. Each well was washed five times with 260 µL of washing buffer, 100 µL of samples were added and incubated for 30 min. Each well was washed five times with 260 µL of washing buffer, 100 µL of diluted horseradish peroxidase supplied in the kit were added, and the plate was incubated for 1 h. Each well was washed five times with 260 µL of washing buffer, 100 µL of tetramethylbenzidine substrate were added, and the plate was incubated in the dark for 15 min. Sulfuric acid (100 µL) at 0.18 M was used as stop solution. The plate was read at 450 nm.
For measurement of total glutathione, a different protein extraction method was used, as recommended by the kit manufacturer total glutathione (STA-312, Cell Biolabs, Inc., San Diego, CA, USA). Mid-jejunum mucosa (100 mg) and 1 mL of metaphosphoric acid at 5% were mixed and homogenized with a glass pestle. The homogenate was centrifuged at 64,000× g for 15 min. The supernatant was used for total glutathione determination total glutathione (STA-312, Cell Biolabs, Inc., San Diego, CA, USA). Glutathione reductase solution (25 µL), NADPH solution (25 µL) supplied in the kit, and samples (100 µL) were added to each well. The chromogen solution (100 µL) supplied in the kit was added to each well and the plate was read at 405 nm every 2 min during 10 min. All wavelengths (for quantifications of protein, protein carbonyls, malondialdehydes, total glutathione, TNF-α, IL-8, IgA, and IgG) were read at the same microplate reader (Synergy HT, Biotek, Winooski, VT, USA).
Ileal digesta was freeze dried (SP Scientific, Virtis 24DX48 GPFD/300820, Warminster, PA, USA) and ground. Subsamples of ground material were analyzed for apparent ileal digestibility of dry matter [90 (link)], gross energy (6200 Calorimeter, Parr Instrument Company, Moline, IL, USA), nitrogen (method 990.03, [91 ], ATC Scientific, North Little Rock, AR, USA), and ether extract (method 920.39, [91 ]).
Fixed mid-jejunal tissue was removed from 10% buffered formaldehyde after two weeks for the obtainment of two transversal cuts that were transferred histological cassettes and submerged in 70% ethanol. Mid-jejunal cuts were included in paraffin for assembling histological slides after staining for Ki-67 antigen. The immunohistochemistry staining with Ki-67 primary monoclonal antibody (1:500 dilution) followed by anti-mouse secondary antibody (1:2 dilution factor) and the use of diamino-benzamine reagent for color development was performed in accordance with methods previously described by Kim et. al. [20 (link)]. Ten pictures of each pig were used to measure gut morphology by a single researcher choosing a well-oriented villus and its associated crypt. Measurements included: villus width (at half of villus height), villus height (from tip of the villus to top of the crypt), crypt depth (from top to bottom of the crypt), and calculating villus height: crypt depth [86 (link)]. The proportion of proliferating cells in the crypt was also estimated by calculating the proportion of cells positive to Ki-67 after taking pictures at 40× in Sony Van–Ox S microscope (Opelco, Washington, DC, USA) and processing in ImageJS tool [92 (link)] for analysis as described by Holanda and Kim [86 (link)].
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Equal amounts (10-30 mg) of protein extracts from the left lung were loaded and separated by SDS-PAGE using 8-10% acrylamide gradients. Following electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific proteins were blocked by incubating the membrane in blocking buffer (5% nonfat dry milk in T-TBS containing 0.05% Tween 20) overnight. The membranes were incubated with monoclonal antibodies against vascular cell adhesion molecule (VCAM)-1 (1: 100, Abcam, Cambridge, MA, USA), intercellular adhesion molecule (ICAM)-1 (1: 2000, Abcam, Cambridge, MA, USA), NAD(P)H quinone oxidoreductase (NQO)-1 (1: 1000, Abcam, Cambridge, MA, USA), connexin43 (Cx43) (1: 2000, Chemicon, Billerica, MA, USA), cytochrom C (Cyt C) (1: 2000, BD, San Jose, CA, USA) and heme oxygense (HO)-1 (1: 250, Abcam, Cambridge, MA, USA), and polyclonal antibodies against TNF-α (1: 1000, Cell Signaling, Danvers, MA, USA) and NFκB (1: 250, Abcam, Cambridge, MA, USA). Signals were detected with horseradish peroxidase (HRP)-conjugated goat anti- mouse, goat anti-rat, or goat anti-rabbit IgG.
The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The procedure of 2,4-dinitrophenylhydrazine (DNPH) derivatization was carried out on 6 μg of protein for 15 minutes according to manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for two hours, followed by incubation with secondary antibody solution (1:300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes.
Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel.
The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The procedure of 2,4-dinitrophenylhydrazine (DNPH) derivatization was carried out on 6 μg of protein for 15 minutes according to manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for two hours, followed by incubation with secondary antibody solution (1:300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes.
Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel.
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Serum Cu and Zn were detected by atomic absorption spectrophotometer (AA26501, Shimadzu, Japan), serum vitamin E by high performance liquid chromatography (HPLC2996 photodiode array detector, Waters, Ltd, USA), serum vitamin C by 2, 4-Dinitrophenylhydrazine Colorimetry (Du2650 ultraviolet spectrophotometer, Beckman, USA), total superoxide dismutase (SOD) and Cu and Zn-SOD by the xanthine oxidase method, and malondialdehyde (MDA) by the 2-thiobarbituric acid (TBA) method. Total antioxidant capacity (TAC) was detected by a colorimetry kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
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Antioxidants
Ascorbic Acid
Colorimetry
dinitrophenylhydrazine
High-Performance Liquid Chromatographies
Malondialdehyde
Serum
Superoxide Dismutase
thiobarbituric acid
Vitamin E
Xanthine Oxidase
Most recents protocols related to «Dinitrophenylhydrazine»
Membrane protein carbonyls were assessed with 2,4-dinitrophenylhydrazine according to Levine et al. [55 (link)].
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Reagent grade 5-chloroisatin, and 2,4-dinitrophenylhydrazine were purchased from Sigma-Aldrich. Ethanol as solvent and concentrated acetic acid were used as purchased.
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Biochemical estimations were carried out for all the samples. Estimation of ascorbic acid done by 2–4 dinitrophenylhydrazine method. The Principle of this method is that Dehydroascorbic acid was coupled with 2, 4 dinitrophenylhydrazine and the resulting derivative is treated with sulphuric acid to produce a newly observed color which is measured at 545 nm.[13 (link)] Iron estimation was done by Ferrozine method and the principle of transferrin bound iron breaks into free ferric ions in an acidic medium. These ferric ions react with Hydroxylamine Hydrochloride reduced into ferrous ions which react with Ferrozine to form a violet colored complex measured at 560 nm. The difference before and after the addition of ferrozine is proportional to iron concentration reaction in the specimen.[14 ]
Protein carbonylation (PC) levels were measured by the quantification of carbonyl groups by using the 2,4-Dinitrophenylhydrazine (DNPH) alkaline method [65 (link)] with adaptations [66 (link)]. Absorbance was read at 450 nm, and the concentration of carbonyl groups was expressed in nmol/mg of protein, by using 22.308 M−1 cm−1 as the molar extinction coefficient of the carbonyl-dinitrophenylhydrazine adduct [65 (link)].
Lipid peroxidation (LPO) levels were determined by the quantification of thiobarbituric acid reactive substances (TBARSs), which are formed in the reaction between LPO by-products (such as malondialdehyde—MDA) and 2-thiobarbituric acid (TBA). Absorbance was read at 532 nm, and the results were calculated by using the molar extinction coefficient of MDA (ε = 1.56105 M−1 cm−1) and expressed in nmol/mg of protein [67 ].
Lipid peroxidation (LPO) levels were determined by the quantification of thiobarbituric acid reactive substances (TBARSs), which are formed in the reaction between LPO by-products (such as malondialdehyde—MDA) and 2-thiobarbituric acid (TBA). Absorbance was read at 532 nm, and the results were calculated by using the molar extinction coefficient of MDA (ε = 1.56105 M−1 cm−1) and expressed in nmol/mg of protein [67 ].
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The performance parameters
of RoLAAO was monitored using the 2,4-dinitrophenylhydrazine
chromogenic method. The principle is that the products α-keto
acids and 2,4-dinitrophenylhydrazine will undergo a color development
reaction to produce brownish-red 2,4-dinitrophenylhydrazone.
of RoLAAO was monitored using the 2,4-dinitrophenylhydrazine
chromogenic method. The principle is that the products α-keto
acids and 2,4-dinitrophenylhydrazine will undergo a color development
reaction to produce brownish-red 2,4-dinitrophenylhydrazone.
Top products related to «Dinitrophenylhydrazine»
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2,4-dinitrophenylhydrazine is a chemical compound used as a reagent in various analytical and organic chemistry applications. It is a yellow crystalline solid that is commonly used for the identification and detection of carbonyl compounds, such as aldehydes and ketones. The compound reacts with these carbonyl groups to form a characteristic hydrazone product, which can be observed and analyzed.
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The OxyBlot Protein Oxidation Detection Kit is a laboratory tool used to detect and analyze oxidative modifications in proteins. It provides a method for the identification and quantification of carbonyl groups introduced into protein side chains as a result of oxidative processes.
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Thiobarbituric acid is a chemical compound used in various laboratory applications. It is a white to pale yellow crystalline solid that is soluble in water and organic solvents. Thiobarbituric acid is commonly used as a reagent in analytical techniques to detect the presence of certain compounds, particularly those related to lipid peroxidation.
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Trichloroacetic acid is a colorless, crystalline chemical compound used in various laboratory applications. It serves as a reagent and is commonly employed in analytical chemistry and biochemistry procedures. The compound's primary function is to precipitate proteins, making it a useful tool for sample preparation and analysis.
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2,4-dinitrophenylhydrazine (DNPH) is a chemical reagent used in analytical and organic chemistry applications. It is a yellow crystalline solid that is commonly used for the detection and identification of carbonyl compounds, such as aldehydes and ketones. DNPH forms a characteristic yellow-orange derivative when reacted with these compounds, which can be used for quantitative analysis.
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Trichloroacetic acid (TCA) is a chemical compound commonly used in analytical and research applications. It is a colorless, crystalline solid that is soluble in water and organic solvents. TCA is a strong acid and is often used as a precipitating agent for proteins and other biomolecules in various laboratory procedures.
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More about "Dinitrophenylhydrazine"
Dinitrophenylhydrazine (DNPH) is a versatile chemical reagent widely used in organic synthesis and analytical chemistry.
It reacts with carbonyl compounds, such as aldehydes and ketones, to form colored hydrazone derivatives, enabling the identification and quantification of these compounds.
This reaction is a valuable tool for researchers, allowing them to detect and analyze a variety of carbonyl-containing molecules.
Closely related to dinitrophenylhydrazine is 2,4-dinitrophenylhydrazine (2,4-DNPH), which is often used in similar applications.
The OxyBlot Protein Oxidation Detection Kit is a commercial product that utilizes 2,4-DNPH to detect and quantify oxidized proteins, a process that is important for understanding cellular stress and oxidative damage.
Bovine serum albumin (BSA) is another commonly used compound in research involving dinitrophenylhydrazine and related techniques.
BSA is often used as a protein standard, and can be used in conjunction with DNPH-based assays to quantify protein content and oxidation levels.
Thiobarbituric acid (TBA) and trichloroacetic acid (TCA) are also relevant compounds in this context.
TBA is used in the thiobarbituric acid reactive substances (TBARS) assay, which can be used to measure lipid peroxidation, a process that can be detected using DNPH-based methods.
Researchers can leverage the innovative PubCompare.ai platform to streamline their workflow when working with dinitrophenylhydrazine and related compounds.
This AI-powered tool allows users to locate relevant protocols from literature, preprints, and patents, and use AI-driven comparisons to identify the best methodologies and products.
This enhances reproducibility and research accuracy, ultimately improving the quality and efficiency of studies involving dinitrophenylhydrazine and related techniques.
It reacts with carbonyl compounds, such as aldehydes and ketones, to form colored hydrazone derivatives, enabling the identification and quantification of these compounds.
This reaction is a valuable tool for researchers, allowing them to detect and analyze a variety of carbonyl-containing molecules.
Closely related to dinitrophenylhydrazine is 2,4-dinitrophenylhydrazine (2,4-DNPH), which is often used in similar applications.
The OxyBlot Protein Oxidation Detection Kit is a commercial product that utilizes 2,4-DNPH to detect and quantify oxidized proteins, a process that is important for understanding cellular stress and oxidative damage.
Bovine serum albumin (BSA) is another commonly used compound in research involving dinitrophenylhydrazine and related techniques.
BSA is often used as a protein standard, and can be used in conjunction with DNPH-based assays to quantify protein content and oxidation levels.
Thiobarbituric acid (TBA) and trichloroacetic acid (TCA) are also relevant compounds in this context.
TBA is used in the thiobarbituric acid reactive substances (TBARS) assay, which can be used to measure lipid peroxidation, a process that can be detected using DNPH-based methods.
Researchers can leverage the innovative PubCompare.ai platform to streamline their workflow when working with dinitrophenylhydrazine and related compounds.
This AI-powered tool allows users to locate relevant protocols from literature, preprints, and patents, and use AI-driven comparisons to identify the best methodologies and products.
This enhances reproducibility and research accuracy, ultimately improving the quality and efficiency of studies involving dinitrophenylhydrazine and related techniques.