Protein Carbonyl content is the most common indicator of protein oxidation. The levels of protein carbonyl in platelets were determined using a Protein carbonyl content Assay Kit (Abcam, US) and all the procedures were performed as per the manufacturer's instruction. Results are expressed as nmol carbonyl/mg protein.
Protein carbonyl content assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Protein Carbonyl Content Assay Kit is a quantitative colorimetric assay that measures the total protein carbonyl content in biological samples. The kit provides a straightforward method to determine the level of oxidative stress in cells or tissues.
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28 protocols using protein carbonyl content assay kit
1
Quantifying Protein Oxidation in Platelets
2
Quantifying Protein Carbonyl Oxidation
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The Protein Carbonyl Content Assay Kit (Abcam) was employed to elicit protein carbonyl groups according to the manufacturer's prescribed guidelines. Tumor tissues were homogenized in deionized water (dH2O) with the aid of a homogenizer. Subsequent to centrifugation, the resulting supernatant was collected, and the samples were diluted with dH2O to achieve an approximate protein concentration of 10 mg/mL. For each individual sample, 100 μL of DNPH was introduced, followed by vigorous mixing and an incubation period of 10 min at room temperature. Subsequently, 30 μL of Trichloroacetic Acid Solution/TCA was added to each sample, followed by further mixing, placement on ice for 5 min, and centrifugation at maximum velocity for 2 min. The supernatant was meticulously extracted and discarded. Cold acetone (500 μL) was utilized to wash the resulting pellet in each tube, and the sample underwent sonication in a sonicating bath for 30 s. Afterward, the sample was placed at −20 °C for 5 min, subjected to a 2-min centrifugation, and the acetone was cautiously removed. Finally, 200 μL of guanidine solution was introduced to each tube and briefly subjected to sonication. The optical density at approximately 375 nm was measured employing a microplate reader within a 96-well plate.
3
Green Tea Extract Bioactive Profiling
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The green tea extract (GTE), Theaphenon E®, kindly provided by Dr. Yukihiko Hara, contained 70% epigallocatechin gallate (EGCG), 5% epigallate catechins (EGC), 4% epicatechin (EC), and 0.6% gallocatechin (GC) [5 (link)]. Sodium hyaluronate (1%) (DuoVisc®) was obtained from Alcon-Couvreur (Puurs, Belgium). Total protein assay was obtained from PierceTM BCA Protein Assay Kit (Rockford, IL, USA). The TUNEL assay kit was purchased from ABCAM (Waltham, MA, USA). The Rat Caspase-3 ELISA Kit was obtained from CUSABIO (Houston, TX, USA). The Protein Carbonyl Content Assay Kit was purchased from ABCAM (Waltham, MA, USA). The Rat MCP-1 instant ELISATM Kits were obtained from InvitrogenTM (Vienna, Austria). The Rat XL Cytokine Array Kit of Proteome ProfilerTM Array was purchased from the R&D system (McKinley Place, MN, USA). Fatty acid methyl ester C4–C24 standard mixture and BSTFA+TMCS (99:1) were obtained from Supelco (Bellefonte, PA, USA). Cholesterol, Cholesterol-2,3,4-13C3, and palmitic acid-d31 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Decanoic acid-d19 was obtained from Cambridge Bioscience (Cambridge, UK). Trimethylsilydiazomethane was purchased from Alfa Aesar (Lancashire, UK). All other reagents were of the highest grade available.
4
Retinal Protein Carbonyl Quantification
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Six retinae from each group were processed following the protocol of the Protein Carbonyl Content Assay Kit (ABCAM, Waltham, MA, USA). Retinae were homogenized in water and centrifuged. Samples were diluted with water to a concentration of 0.5–2 mg in 100 μL protein per assay. DNPH (100 μL) was added to each sample, vortexed, and incubated for 10 min at room temperature. TCA (30 μL) was mixed with each sample, vortexed, placed on ice for 5 min, and then centrifuged for 2 min. The supernatant was discarded without disturbing the pellet. The pellet was suspended with cold acetone and washed. After sonication to disperse the pellets, they were incubated at −20 °C for 5 min, centrifuged for 2 min, and the acetone was removed. Guanidine solution (200 μL) was added, and the mixture was sonicated to solubilize the proteins. After a brief spin to remove insolubilized material, each sample (100 μL) was transferred to a 96-well plate and measured at approximately 375 nm.
5
Protein Carbonyl Quantification in Neurodegeneration
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Protein carbonyl levels were measured utilizing the Protein Carbonyl Content Assay Kit (Abcam, Cambridge, MA, United States) according to manufacturer’s protocol. Briefly, protein was extracted from the neocortex of animals nasally administered IFNγ-DC-Exos or sodium succinate vehicle (sham) using RIPA buffer. Protein homogenate was treated with streptozocin to remove any nucleic acid contaminates. Samples were reacted with 2, 4-Dinitrophenylhydrazine followed by quantification of the acid hydrazones at 405 nm. BCA assays (Thermo Fisher Scientific) were simultaneously run and a standard curve constructed for the calculation of protein carbonyl content based on optical density.
6
Oxidative Stress Measurement in Lung
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The methods for measuring the MDA level and protein carbonyl content were described previously (16 (link)). The protein carbonyl contents and MDA levels in the upper lobe of the right lung were determined using a Protein Carbonyl Content Assay Kit (Abcam, USA) and an MDA Assay Kit (Abcam), respectively, according to the manufacturer's instructions. The results of both assays are reported as nmol/mg protein.
7
Protein Oxidation Quantification Protocol
8
Quantifying Oxidative Stress Biomarkers
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The levels of protein carbonyls (PCs) were measured using a commercial Protein Carbonyl Content Assay Kit (Abcam, USA) as previously described [6 (link)]. In brief, fresh lung samples were homogenized, treated with streptozocin to remove nucleic acids, and incubated with DNPH (100 μL), TCA (30 μL), cold acetone (500 μL), and guanidine solution (200 μL) following the manufacturer's instructions. Then, PCs were measured spectrophotometrically at 375 nm and expressed as pmol/mg protein. The contents of 3-nitrotyrosine (3-NT), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) were also detected to assess protein and lipid peroxidation using commercial kits (Abcam, USA). 8-hydroxy 2 deoxyguanosine (8-OHdG) is produced by oxidative DNA damage, and 8-OHdG levels in fresh lung homogenates were evaluated using an 8-OHdG-coated plate and an HRP-conjugated antibody according to the manufacturer's instructions (Abcam, USA). The absorbance was measured at 450 nm and used to calculate total protein concentrations as previously described.
9
Quantifying Protein Thiols and Oxidation
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The Ellman’s method was used to determine the content of free thiols [31 (link)]. Mouse serum albumin (MSA, UniProtKB P07724) and rMSA were mixed with equal volumes of 5, 5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) reagent, respectively. The volume and concentration of DTNB used in this study were 100 μL and 2 mM, respectively. Then, 800 μL Tris buffer (1 M) was added to make the volume of the reaction system reach 1000 μL. Samples were kept at room temperature for 30 min. The fluorescence absorbance was measured at 412 nm. Carbonyls in protein samples were quantified using the Protein Carbonyl Content Assay Kit (Abcam, Cambridge, UK, ab126287) according to the manual. Hcy concentrations were measured by the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (MEIMIAN, Yancheng, China, 1213). Concentrations of AGE were measured with an ELISA kit according to manufacturer’s instructions (CLOUD-CLONE Co., Wuhan, China, CEB353Ge).
10
Protein Carbonylation in HEK Cells
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HEK cells (4 × 106 cells/well) were seeded onto 6-well plates, and the cells were incubated with vehicle (control) or 40 μM DPHC for 1 h and then further incubated with or without 1 mM MGO for 24 h or 20 mM glucose for 30 h. Carbonylation of protein causing oxidative damage was measured in HEK cells using a Protein Carbonyl Content Assay Kit (Abcam, Cambridge, UK) according to the manufacturer's instructions.
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