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    products/assay-kits/protein-carbonyl-assay-kit-western-blot-ab178020.pdf

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Kits/ Lysates/ Other Tools and Reagents Western Blot Tools and Reagents Detection Oxidized protein
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Protein Carbonyl Assay Kit (Western Blot) (ab178020)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (2) Submit a question References (46)

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Sensitivity of DNP
  • Example of DNP detection

Key features and details

  • Sample type: Adherent cells, Suspension cells, Tissue Homogenate

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Overview

  • Product name

    Protein Carbonyl Assay Kit (Western Blot)
  • Sample type

    Adherent cells, Suspension cells, Tissue Homogenate
  • Product overview

    Protein Carbonyl Assay Kit (Western Blot) ab178020 is designed for the measurement of protein carbonyl groups that are created by the oxidation of proteins. This can be through reaction with ozone or oxides of nitrogen, or by metal catalyzed oxidation.


    In the protein carbonyl assay protocol, carbonyl groups in protein side chains are derivatized to DNP-hydrazone by reaction with DNPH. The DNP moieties are detected using an anti-DNP antibody and western blotting.


    Using an immunoblotting method for the protein carbonyl assay has the advantage that individual oxidized proteins are separated and identified from a complex mixture by SDS-PAGE. The oxidative status of each protein can be compared between samples.


    Protein carbonyl assay protocol summary:
    - extract proteins from samples with extraction buffer, 20 min incubation, centrifugation at 18,000 x g for 20 min, and retention of the supernatant
    - denature proteins with SDS solution
    - incubate with DNPH for 15 min
    - add neutralization solution
    - run samples on polyacrylamide gel
    - transfer proteins to membrane
    - block for 1 hr
    - incubate with DNP antibody for 3 hr, and wash
    - incubate with HRP goat anti-rabbit secondary antibody for 1 hr, and wash
    - develop with ECL and acquire image

  • Notes

    Previously called Oxidized Protein Assay Kit (Western blot).

    Oxygen-derived free radicals have been implicated in important roles in aging, apoptosis, and cancer. These highly reactive chemical species are also involved in a wide variety of clinical disorders, such as atherosclerosis, cataractogenesis, neurodegenerative diseases, chronic inflammatory diseases, pulmonary diseases and cardiovascular diseases.

    Related products

    Other Protein Carbonyl Content assays include:
    - Protein Carbonyl Content Assay (DNPH) ab126287 (most popular protein carbonyl content assay)
    - Protein Carbonyl Content Assay (Fluorometric) ab235631
    - Protein Carbonyl Content ELISA (DNPH) ab238536

    Review the oxidative stress marker and assay guide to learn about more assays for oxidative stress.

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 100 tests
    12%SDS 1 x 2ml
    1X 2,4-Dinitrophenylhydrazine (DNPH) solution 1 x 4ml
    1X Derivatization Control Solution 1 x 4ml
    2X Extraction Buffer 1 x 4ml
    5000X HRP Conjugated Scondary Antibody 1 x 100µl
    5000X Primary anti-DNP Antibody 1 x 100µl
    Neutralization Solution 1 x 4ml
    Standard protein with DNP residues 1 x 1ml
  • Research areas

    • Kits/ Lysates/ Other
    • Tools and Reagents
    • Western Blot Tools and Reagents
    • Detection
    • Oxidized protein

Images

  • Sensitivity of DNP
    Sensitivity of DNP

    Sensitivity of DNP detection demonstrated by serial dilution of DNP –derivatized BSA.

    Lane 1: 5 ng BSA

    Lane 2: 10 ng BSA

    Lane 3: 20 ng BSA

    Lane 4: 40 ng BSA

    Lane 5: 80 ng BSA

    Lane 6: 160 ng BSA

  • Example of DNP detection
    Example of DNP detection

    Example of DNP detection demonstrated by DNP derivatized H2O2 treated Hela cells.
    Lane 1: MW ladder
    Lane 2 (DNPH): HeLa cells with 0.1 mM H2O2, 15 minutes treatment.
    Lane 3 (Negative Control): HeLa cells with 0.1 mM H2O2, 15 minutes treatment.
    Lane 4 (DNPH): HeLa cells with 1 mM H2O2, 15 minutes treatment.
    Lane 5 (Negative Control): HeLa cells with 1 mM H2O2, 15 minutes treatment.
    Lane 6 (DNPH): HeLa cells with 10 mM H2O2, 15 minutes treatment.
    Lane 7 (Negative Control): HeLa cells with 10 mM H2O2, 15 minutes treatment.
    Lane 8 (DNPH): HeLa cells with 0 mM H2O2, 15 minutes treatment.
    Lane 9 (Negative Control): HeLa cells with 0 mM H2O2, 15 minutes treatment.
    Lane 10 (DNPH): BSA with DNPH derivatization.
    Lane 11 (Negative Control): BSA with DNPH derivatization.

     

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (46)

Publishing research using ab178020? Please let us know so that we can cite the reference in this datasheet.

ab178020 has been referenced in 46 publications.

  • Chaouhan HS  et al. Calycosin Alleviates Paraquat-Induced Neurodegeneration by Improving Mitochondrial Functions and Regulating Autophagy in a Drosophila Model of Parkinson's Disease. Antioxidants (Basel) 11:N/A (2022). PubMed: 35204105
  • Fillmore N  et al. Cardiac specific knock-down of peroxisome proliferator activated receptor a prevents fasting-induced cardiac lipid accumulation and reduces perilipin 2. PLoS One 17:e0265007 (2022). PubMed: 35259201
  • Sayles NM  et al. Mutant CHCHD10 causes an extensive metabolic rewiring that precedes OXPHOS dysfunction in a murine model of mitochondrial cardiomyopathy. Cell Rep 38:110475 (2022). PubMed: 35263592
  • Li Y  et al. Hydroxysafflor Yellow A Blocks HIF-1a Induction of NOX2 and Protects ZO-1 Protein in Cerebral Microvascular Endothelium. Antioxidants (Basel) 11:N/A (2022). PubMed: 35453413
  • Chen L  et al. The role of NADPH oxidase 1 in alcohol-induced oxidative stress injury of intestinal epithelial cells. Cell Biol Toxicol N/A:N/A (2022). PubMed: 35639301
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-2 of 2 Abreviews or Q&A

DNP detection in samples with different extraction methods – protein carbonyl Western Blot kit extraction buffer vs TRIzol

Average Good 4/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Oxidised protein Western Blot detection was performed with proteins extracted from pigs’ kidneys.
Lane 1 (DNPH): Sample 1 extracted with kit’s buffer.
Lane 2 (Negative control): Sample 1 extracted with kit’s buffer.
Lane 3 (DNPH): Sample 1 extracted with TRIzol.
Lane 4 (Negative control): Sample 1 extracted with TRIzol.
Lane 5 (DNPH): Sample 2 extracted with kit’s buffer.
Lane 6 (Negative control): Sample 2 extracted with kit’s buffer.
Lane 7 (DNPH): Sample 2 extracted with TRIzol.
Lane 8 (Negative control): Sample 2 extracted with TRIzol.
Lane 9: standard protein with DNP residuals.
Lane 10: protein ladder.

Sample 1 – 15 µg protein loaded.
Sample 2 – 9 µg protein loaded.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Rūta Žulpaitė

Verified customer

Submitted May 03 2021

DPN detection in samples with different buffer extraction

Average Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Oxidized protein Western Blot Detection in samples from rat brain (cerebral cortex and hippocampus).
Lane1 (DNPH): Samples extracted with kit's buffer.
Lane 2 (Negative control): Samples extracted with kit's buffer.
Lane3 (DNPH): Samples extracted with high saline content buffer.
Lane3 (Negative control): Samples extracted with high saline content buffer.
High saline content buffer composition: 100 mM NaCl, 50 mM PBS, 1 mM NaV, 1 mM NaF and proteases inhibitor.
10 µg of protein in each lane.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jul 10 2017

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