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Protein carbonyl assay kit

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The Protein Carbonyl Assay Kit is a colorimetric assay designed to measure the total protein carbonyl content in biological samples. Protein carbonyls are used as a marker of protein oxidation, which can indicate oxidative stress levels in cells or tissues.

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Lab products found in correlation

Gsh gssg glo, by Promega (4 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Precellys 24 homogenizer, by Avantor (1 mentions) Ldh assay kit, by Promega (1 mentions) Lactate assay kit, by Elabscience (1 mentions) Antibodies against gapdh, by Merck Group (1 mentions) Histone h3, by Cell Signaling Technology (1 mentions) Dna damage elisa kit, by StressMarq Biosciences (1 mentions) Ab126287, by Abcam (1 mentions) Glycogen assay kit, by Merck Group (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Vdac1, by Santa Cruz Biotechnology (1 mentions) Dna pkcs, by Santa Cruz Biotechnology (1 mentions) Parkin, by Abcam (1 mentions) β actin, by Merck Group (1 mentions) Microplate reader, by Agilent Technologies (1 mentions)

23 protocols using protein carbonyl assay kit

1

Evaluating Oxidative Stress Biomarkers

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A8-OH-dG level and ratio of GSH/GSSG allows for evaluation of the status of oxidative stress in the biological systems [32 (link)], while protein carbonyl content can measure Oxidative damage to proteins. The tissue lysates of the core and edge of minipig spinal cord glioma samples were harvested and quantified for 8-hydroxy 2 deoxyguanosine Kit (ab201734, ABCAM, USA), GSH/GSSG-Glo™ (Promega Corporation, USA) and Protein Carbonyl Assay Kit (#ab126287, ABCAM, USA) following with manufacturer's assays procedure.
Tora M.S., Neill S.G., Lakhina Y., Assed H., Zhang M., Nagarajan P.P., Federici T., Gutierrez J., Hoang K.B., Du Y., Lei K, & Boulis N.M. (2023). Tumor microenvironment in a minipig model of spinal cord glioma. Journal of Translational Medicine, 21, 667.
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2

Quantifying Protein Carbonyl Oxidation

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RPE monolayers (ARPE-19, ARPE-19 + shDJ-1, and ARPE-19 + shctrl) were lysed as described above. Protein carbonyl residues in lysates were quantified in 10 μg of the cell lysates using the Protein Carbonyl Assay Kit (Abcam, Boston, MA, USA, ab178020). Derivatization and neutralization were carried out following the manufacturer’s protocol. A parallel set of lysates were treated with the 1x Derivatization Control solution. Individual oxidized proteins were resolved by SDS-PAGE on 12.5% polyacrylamide gel gradient gel (BioRad, 5671043). Proteins were transferred to PVDF membrane at 70 V for 2–3 h. The membranes were blocked using 5% skimmed milk in PBS for 2 h and incubated with primary anti-DNP rabbit antibody (1:5000) overnight at 4 °C, followed by incubation with anti-rabbit IRDye® 800CW. The same membranes were next incubated with beta actin antibodies as described above. Signal intensity in the derivatized lanes and the control lanes was normalized with the beta actin signals in the respective lanes.
Bhattacharyya S., Sturgis J., Maminishkis A., Miller S.S, & Bonilha V.L. (2022). Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function. International Journal of Molecular Sciences, 23(17), 9938.
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3

Protein Carbonyl Quantification in Heart

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The levels of protein carbonyl groups were assessed using the Protein Carbonyl Assay Kit (Abcam) according to manufacturer’s instructions. Briefly, 3 mg/mL solubilized heart protein samples were diluted 1:1 with 12% SDS (final concentration 6% SDS) and then incubated with either DNPH or Derivatization Control Solution for 15 minutes at room temperature, then neutralized. Equal amounts of sample and the DNP-BSA positive control were loaded into a 4–12% SDS–PAGE gel without denaturing or heat application. Samples were blocked in 5% BSA TBST followed by overnight incubation at 4◦C with anti-DNP antibody. The following day blots were washed and incubated with 1X HRP conjugated Secondary Antibody at room temperature for 1 hour. Blots were imaged as described above.
Sayles N.M., Southwell N., McAvoy K., Kim K., Pesini A., Anderson C.J., Quinzii C., Cloonan S., Kawamata H, & Manfredi G. (2022). Mutant CHCHD10 causes an extensive metabolic rewiring that precedes OXPHOS dysfunction in a murine model of mitochondrial cardiomyopathy. Cell reports, 38(10), 110475.
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4

Cellular Redox and Protein Oxidation

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GSH/GSSG ratio and Protein carbonyl levels were measured from cell homogenates using GSH/GSSG-Glo™ (Promega Corporation, USA) and Protein Carbonyl Assay Kit (#ab126287, Abcam, Cambridge MA, USA), respectively.
Lei K., Xia Y., Wang X.C., Ahn E.H., Jin L, & Ye K. (2020). C/EBPβ mediates NQO1 and GSTP1 anti-oxidative reductases expression in glioblastoma, promoting brain tumor proliferation. Redox Biology, 34, 101578.
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5

Protein Carbonylation Assay Protocol

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Protein carbonylation was assessed using the Protein Carbonyl Assay Kit (ab178020, Abcam, Cambridge, MA, USA). Briefly, homogenates from freshly dissected lenses from 1- and 2.5-month-old Cx50D47A mice and 2.5- and 4.5-month-old Cx46fs380 mice were prepared, and the protein concentrations were determined as described in Section 2.2. Equal amounts of proteins from each lens homogenate were incubated with 2,4-dinitrophenylhydrazine to modify the carbonyl groups in the proteins with a 2,4-dinitrophenyl (DNP) group. The derivatized proteins were resolved by SDS-PAGE using 15% polyacrylamide gels and electrotransferred onto Immobilon P. The membranes were incubated with a 1:5000 dilution of the rabbit anti-DNP antibodies followed by a 1:5000 dilution of the horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies provided in the kit. Densitometric analyses were performed using Adobe Photoshop CS3 by drawing a rectangular box encompassing the entire lane.
Jara O., Minogue P.J., Berthoud V.M, & Beyer E.C. (2020). Do Connexin Mutants Cause Cataracts by Perturbing Glutathione Levels and Redox Metabolism in the Lens?. Biomolecules, 10(10), 1418.
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6

Oxidative Stress Biomarkers Quantification

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After different treatments, the protein carbonyl level and GSH/GSSG ratio were measured from cell homogenates using a Protein Carbonyl Assay Kit (#ab126287, Abcam, Cambridge, MA, USA) and GSH/GSSG-Glo™ (Promega Corporation, USA), respectively, according to the manufacturer's guidelines [33 (link)]. All tests were performed in triplicate.
Lei K., Shen Y., He Y., Zhang L., Zhang J., Tong W., Xu Y, & Jin L. (2020). Baicalin Represses C/EBPβ via Its Antioxidative Effect in Parkinson's Disease. Oxidative Medicine and Cellular Longevity, 2020, 8951907.
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7

Quantification of Protein Carbonylation

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Carbonyl groups in oxidized proteins were detected by the Protein Carbonyl Assay Kit (ab178020, Abcam). In brief, aortic tissue (30–50 mg) was homogenized in 1 mL of DPBS supplemented with 1:100 Halt Protease and Phosphatase inhibitor using a Precellys 24 homogenizer (VWR). Afterwards, 50 µL of extraction buffer was added to the aortic homogenate and isolation was performed according the manufacturer's instructions. Protein concentrations were determined by bicinchoninic acid assay (BCA) and afterwards, 50 mmol/L dithiothreitol (DTT) was added to the samples. Proteins (4 µg/µL) were separated by 4% to 12% Bis‐Tris Protein Gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes. Incubation with primary and secondary antibodies was performed according to the manufacturer's instructions. Negative controls were run in pretests (Figure S2) and an internal control was run on every gel. Data were normalized to this control, which was set to =1.
Hofmann A., Müglich M., Wolk S., Khorzom Y., Sabarstinski P., Kopaliani I., Egorov D., Horn F., Brunssen C., Giebe S., Hamann B., Deussen A., Morawietz H., Poitz D.M, & Reeps C. (2021). Induction of Heme Oxygenase‐1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(20), e022747.
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8

Protein Carbonyl Quantification

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The amount of protein carbonyl groups was measured with the Protein carbonyl assay kit (Abcam). Proteins were quantified by Bradford.
Thelen M.P., Wirth B, & Kye M.J. (2020). Mitochondrial defects in the respiratory complex I contribute to impaired translational initiation via ROS and energy homeostasis in SMA motor neurons. Acta Neuropathologica Communications, 8, 223.
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9

Oxidative Stress Response to Bacillus Subtilis

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The level of oxidized protein was measured with the Oxyblot kit (Protein Carbonyl Assay Kit, Abcam) according to the manufacturer protocol. Trophozoites (1 × 106) were incubated for 1 h at 37 °C with B. subtilis planktonic form, B. subtilis biofilm form or with serum-free Diamond’s TYI S-33 medium without bacteria (as control) in 24-well plates cover with serum-free Diamond’s TYI S-33 agar. Then, the cells were exposed to H2O2 (2.5 mM, for 30 min at 37 °C). Trophozoites were then lysed with Nonidet P-40 (NP-40 1% in DDW) for 15 min on ice. Equal protein concentrations (20 μg) were proceeded with the oxyblot, protein oxidation detection kit.
Zanditenas E., Trebicz-Geffen M., Kolli D., Domínguez-García L., Farhi E., Linde L., Romero D., Chapman M., Kolodkin-Gal I, & Ankri S. (2023). Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity. NPJ Biofilms and Microbiomes, 9, 77.
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10

Detecting Protein Carbonylation in hiPSC-CM

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Detection of protein carbonylation in PLNic and PLN p. Arg14del hiPSC‐CM was assessed using the Protein Carbonyl Assay Kit (Abcam #ab178020) according to the manufacturer’s instructions. In brief, 2D hiPSC‐CMs were washed with PBS and harvested in extraction buffer containing 50 mM DTT. Proteins were denatured by SDS and incubated with 2,4‐dinitrophenylhydrazine (DNPH) or control derivatization solution for 15 min. The reaction was stopped by addition of neutralization buffer. Protein samples were separated by 10% SDS–PAGE, blotted onto nitrocellulose, and blocked in 10% non‐fat milk in TBS‐T. Membranes were probed with an anti‐DNP antibody (1:5,000 in blocking buffer). Equal protein loading between DNPH and control‐derivatized samples was ascertained by probing with an antibody against cMyBP‐C. Proteins that underwent oxidation were detected by the anti‐DNP antibody, only in the DNPH‐derivatized samples.
Cuello F., Knaust A.E., Saleem U., Loos M., Raabe J., Mosqueira D., Laufer S., Schweizer M., van der Kraak P., Flenner F., Ulmer B.M., Braren I., Yin X., Theofilatos K., Ruiz‐Orera J., Patone G., Klampe B., Schulze T., Piasecki A., Pinto Y., Vink A., Hübner N., Harding S., Mayr M., Denning C., Eschenhagen T, & Hansen A. (2021). Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation. EMBO Molecular Medicine, 13(6), e13074.
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