Rat gastrocnemius homogenates were analyzed for advanced oxidative protein products using the OxiSelect AOPP Assay Kit (kit # STA-318, Cell Biolabs, Inc., San Diego, CA, USA). Samples were diluted 1:20 with the assay diluent included in the kit and 200 µl of the prepared samples were added per well to a 96-well plate. Chloramine reaction initiator (provided in the kit) was added to each well and the plate incubated for 5 min at room temperature with shaking at 250 rpm. The reaction was then terminated, and the plate read at 340 nm using the BioTek Synergy Model H1M Multimodal Plate Reader. The AOPP concentrations in the gastrocnemius homogenates were determined by comparing to a standard curve prepared using Chloramine-T.
Oxiselect aopp assay kit
Manufactured by Cell Biolabs
Sourced in United States
The OxiSelect AOPP Assay Kit is a colorimetric assay designed to quantify advanced oxidation protein products (AOPPs) in biological samples. The kit provides reagents and protocols to measure the concentration of AOPPs, which are protein markers of oxidative stress.
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Lab products found in correlation
Zenyth 200 microplate spectrophotometer, by Anthos Labtec (3 mentions)
Synergy model h1m multimodal plate reader, by Agilent Technologies (2 mentions)
Oxiselect total antioxidant capacity assay kit, by Cell Biolabs (2 mentions)
Protein carbonyl colorimetric assay kit, by Cayman Chemical (1 mentions)
Hypochlorous acid, by Fluke (1 mentions)
Limulus amoebocyte lysate kit, by Lonza (1 mentions)
Oxiselect hne adduct competitive elisa kit, by Cell Biolabs (1 mentions)
Oxiselect age competitive elisa kit, by Cell Biolabs (1 mentions)
Human rage quantikine elisa kit, by R&D Systems (1 mentions)
Tbars assay kit, by Cayman Chemical (1 mentions)
14 protocols using oxiselect aopp assay kit
1
Oxidative Stress Assessment in Rat Muscle
2
Oxidative Stress Biomarker Quantification
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AOPP (OxiSelect AOPP Assay Kit; Cell Biolabs, Inc., USA), protein carbonyl content, and total protein content (Protein Carbonyl Colorimetric Assay Kit; Cayman Chemical Company, USA) analyses were performed according to the methods published previously by Gryszczyńska et al. [11 (link)]. Shortly, the chloramine reaction initiator was used to measure the concentration of AOPP, which was expressed per total protein content (μmol/mg protein). The concentration of C=O was measured based on the reaction between protein carbonyls and 2,4-dinitrophenylhydrazine (DNPH), whereas dilution of proteins with guanidine hydrochloride followed by measurement of the absorbance at 280 nm was used to determine the total protein content. Colorimetric assay kits were carried out using the Zenyth 200 Microplate Spectrophotometer (Anthos Labtec Instruments GmbH).
3
Quantifying Oxidative Protein Damage
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Serum samples were analyzed for advanced oxidative protein products using the OxiSelect AOPP Assay Kit (kit # STA-318, Cell Biolabs, Inc., San Diego, CA, USA). Serum samples were diluted to 1 : 20 with the assay diluent included in the kit, and 200 μl of the prepared samples was added per well to a 96-well plate. A chloramine reaction initiator, provided in the kit, was added to each well and the plate incubated for 5 min at room temperature with shaking (250 rpm). The reaction was then terminated and the plate read at 340 nm using the BioTek Synergy Model H1M Multimodal Plate Reader. The AOPP concentrations in the serum samples were determined by comparing to a standard curve prepared using chloramine-T. The lower limit of detection is approximately 1.25 μM.
4
In Vitro Preparation and Quantification of AOPP
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AOPP were prepared in vitro by incubating rate serum albumin (RSA) (Sigma, St. Louis, MO, USA) with hypochlorous acid (Fluke, Buchs, Switzerland) as previously described [22] (link). AOPP in the preparations were determined with an OxiSelect AOPP Assay Kit (Cell Biolabs, San Diego, CA, USA). The content of AOPP was 50.10 ± 3.92 μmol/g protein in AOPP, and 0.22 ± 0.06 μmol/g protein in unmodified rate serum albumin (RSA).
5
In vitro Preparation of AOPP-RSA
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AOPP-RSA was prepared in vitro by incubation of RSA (Sigma, St. Louis, MO, USA) with HOCl (Fluke, Buchs, Switzerland) as described previously.10 (link) Prepared samples were dialyzed against phosphate-buffered saline (PBS) for 24 h to remove free HOCl and passed through a Detoxi-Gel column (Pierce, Rockford, IL, USA) to remove contaminated endotoxin. Endotoxin levels in AOPP-RSA were measured with a Limulus Amoebocyte Lysate kit (BioWhittaker, Walkersville, MD, USA) and were found to be below 0.05 ng/mg protein. AOPPs contents in the preparations were determined with an OxiSelect AOPP Assay Kit (Cell Biolabs, San Diego, CA, USA); AOPPs contents in the AOPP-RSA and unmodified RSA were 50.10±3.92 and 0.22±0.06 μmol/g protein, respectively.
6
Quantification of Oxidative Stress Marker AOPP
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Advanced oxidation protein products (AOPPs), a marker of oxidative stress, were measured in BALF of mice using the OxiSelect AOPP assay kit (STA-318; Cell Biolabs, San Diego, CA, USA) following the manufacturer’s instructions. The AOPP content was determined by comparison with a chloramine standard curve and expressed in micromolar concentration chloramine equivalent units.
7
Oxidative Stress Biomarkers Quantification
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Protein oxidation was measured using OxiSelect™ AOPP Assay Kit (Cell Biolabs Inc., San Diego, CA, USA) for advanced oxidation protein products (expressed as μmol of chloramine equivalents (CE)/mg of tissue protein). Lipid peroxidation had been evaluated by OxiSelect™ HNE Adduct Competitive ELISA Kit (Cell Biolabs Inc.) for hydroxynonenal (HNE) protein adducts (expressed as μg/mL of tissue homogenate). Total antioxidant capacity was assessed by OxiSelect™ Total Antioxidant Capacity (TAC) Assay Kit (Cell Biolabs Inc.) expressed as μmol of copper reducing equivalents (CRE)/mg of tissue.
8
Oxidative Stress Quantification
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Circulating oxidative stress was assayed using Cell Biolabs, Inc., OxiSelect AOPP assay kit, according to our previously published protocol (Cunningham et al. 2011 ). This kit measures the amount (μmol/L) of all oxidized proteins in the sample relative to a known standard. Chloramine in the kit reacts with oxidized proteins to produce a color change which can be read at 340 nm.
9
Quantification of Oxidative Stress Marker AOPP
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Advanced oxidation protein products (AOPPs), a marker of oxidative stress, were measured in BALF of mice using the OxiSelect AOPP assay kit (STA-318; Cell Biolabs, San Diego, CA, USA) following the manufacturer’s instructions. The AOPP content was determined by comparison with a chloramine standard curve and expressed in micromolar concentration chloramine equivalent units.
10
Evaluating Plasma and CSF Biomarkers
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At the termination of the experiment, rats were euthanized, and the blood was collected into prechilled 10-ml tubes containing EDTA. Plasma was separated by centrifugation for 15 min at 3000 rpm at 4°C. The levels of TMAO in plasma and CSF, the levels of norepinephrine (NE, a marker of sympathetic nerve activity), and the levels of proinflammatory cytokines IL-1β and IL-6 in plasma were determined with commercially available ELISA kits (MyBioSource, Inc. San Diego, CA, United States; Rocky Mountain Diagnostics, Inc., Colorado Springs, United States; Thermo Fisher Scientific, Grand Island, NY, United States). The levels of the advanced oxidation protein products (AOPP, a marker of systemic oxidative stress) in plasma were assessed using an OxiSelect™ AOPP Assay Kit (Cell Biolabs, Inc. San Diego, CA, United States).
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