Protein carbonylation was assessed by measuring the levels of carbonyl groups using the OxyBlot Protein Detection Kit from Millipore, as previously described73 (link). Briefly, 20 μg of soluble proteins were denatured by 6% SDS (w:v) and the carbonyl groups in the protein side chains were derivatized to 2,4-dinitrophenylhydrazone (DNP) by reaction with 2,4-dinitrophenylhydrazine (DNPH). The reaction was stopped and the skeletal muscle protein carbonylation was detected by Western Blotting, quantified in the broadest molecular weight range as possible and expressed as percentage of Control or Sed-SNC groups (arbitrarily set as 100%).
Oxyblot protein detection kit
Manufactured by Merck Group
Sourced in United States
The OxyBlot Protein Detection Kit is a laboratory product used to detect and quantify the levels of oxidatively modified proteins in biological samples. The kit provides the necessary reagents and protocols to perform this analysis.
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9 protocols using oxyblot protein detection kit
1
Protein Carbonylation Quantification
2
Oxidative Stress Biomarkers Analysis
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Protein carbonyls were assessed with the OxyBlot Protein Detection Kit (S7150; Millipore, Switzerland). Soluble proteins (20 μg) were denatured by SDS and derivatized by Dinitrophenylhydrazine (DNPH). Proteins were subjected to electrophoresis and immunoblotting according to the manufacturer's instructions. For dihydroethidium (DHE) fluorescence staining, muscle cross-sections (10 μm) were incubated with DHE (5 μM) in a light-protected incubator at 37 °C for 30 min. The sections were washed with phosphate buffered saline and fluorescence was assessed by confocal microscopy. Quantitative analysis of fluorescent images was performed with ImageJ (NIH, USA). Lipid hydroperoxides were evaluated using the modified ferrous oxidationxylenol (FOX) orange technique [65 (link)], and malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) were assessed using an ALDetect Lipid Peroxidation Assay Kit (Enzo Life Sciences International, NY, USA).
3
Oxidative Protein Carbonylation Quantification
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Oxidative protein carbonylation on tibialis anterior muscle was tested using an OxyBlot Protein Detection Kit (Millipore) according to the manufacturer's instructions. The carbonyl groups in protein side chains were derivatized to DNP-hydrazone with DNPH. The modified extract is then loaded on a 12% SDS-PAGE, transferred on a PVDF membrane and blotted with an anti-DNP antibody. Band intensities were quantified with the ImageJ software and normalized to the total protein content.
4
Glycation and Carbonylation in Flies
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Levels of glycation in whole frozen flies (n = 10 per samples) were assessed by western blotting using an anti-AGE (advanced glycation end-product) antibody (1:2,000; Millipore AB9890), normalized against total protein assessed using the stain-free labeling system (Bio-Rad). Carbonylation was measured using the OxyBlot Protein Detection Kit (Millipore S7150). Whole frozen flies (n = 5 per sample) were homogenized in 100 μL of CelLytic M Cell Lysis Reagent (Sigma C2978) supplemented with 50 mM DTT and protease inhibitor cocktail (complete mini EDTA-free, Roche 11 836 170 001). Protein samples (20 μg, assayed by the Bradford method) were subjected to OxyBlot derivatization according to the manufacturer’s instructions, followed by western blotting. Lanes were analyzed by densitometry using Image Lab software (Bio-Rad).
5
Oxidative Protein Carbonylation Assay
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Oxidative protein carbonylation assays were performed on lung tissue following western blot using an OxyBlot Protein Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The carbonyl groups in the protein side chains were derivatized to DNP-hydrazone by reaction with 2,4-dinitrophenylhydrazine (DNPH) following the manufacturer’s instructions. After derivatization of the protein sample, one-dimensional electrophoresis was carried out on 10% SDS polyacrylamide gel electrophoresis gels. Proteins were transferred to polyvinylidene difluoride membranes. After incubation with an anti-DNP antibody, the blot was developed using a chemiluminescence detection system.28 (link)
6
Oxidative Protein Carbonylation Assay
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The oxidative protein carbonylation assay was performed on lung tissue using an OxyBlot Protein Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. The carbonyl groups in the protein side chains were derivatised to dinitrophenylhydrazine (DNP)‐hydrazone by reaction with 2,4‐DNP. The proteins were then separated by 10% SDS‐PAGE and transferred to a PVDF membrane. After incubation with an anti‐DNP antibody, the protein band was detected with a chemiluminescence system.
7
Oxidative Protein Carbonylation Assay
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Oxidative protein carbonylation assays were performed on tissue following western blot using an OxyBlot Protein Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions28 (link). The carbonyl groups in the protein side chains were derivatized to DNP-hydrazone by reaction with 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH) following the manufacturer’s instructions. After derivatization of the protein sample, one-dimensional electrophoresis was carried out on 10% electrophoresis gels. Proteins were transferred to PVDF membranes. After incubation with an anti-DNP antibody, the blot was developed using a chemiluminescence detection system.
8
Quantifying Oxidative Protein Damage
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Samples of spinal cord, taken from six rats for each group, were homogenized in lysis buffer, as previously described (Tayebati et al., 2017 (link)). We assessed protein carbonylation by treating equal amounts of protein according to protocol of OxyBlot Protein detection kit (Millipore, USA, Cat. No. S7150). The kit provides a system to perform the immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions. As a consequence, carbonyl groups are introduced into the side chains of all proteins independently of the molecular weight. The samples were separated by 8% SDS polyacrylamide gel, transferred onto nitrocellulose and blotted with the specific antibodies of the kit that recognize all the oxidized protein with different molecular weight. Band intensities were measured by densitometry with IAS 2000 image analyzer (Biosystem, Rome, Italy).
9
Protein Carbonyl Quantification by OxyBlot
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Protein carbonyls were assessed by using the OxyBlot Protein Detection Kit (S7150; Millipore, USA). Soluble proteins (20 µg) were denatured by SDS and derivatized by DNPH. Proteins were submitted to electrophoresis and immunoblotting accordingly the manufacturer’s instructions.
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