BioCell Protein Carbonyl Assay Kit

The BioCell Protein Carbonyl Assay Kit is an enzyme-linked immuno-sorbent assay (ELISA) for the measurement of protein carbonyls in biological samples.

Each kit contains everything you need, including standards, control samples, all reagents, detailed instructions and plate.

An extensive overview by Weber and colleagues (Redox Biology 5 (2015) 367–380) of different methods concluded that “ELISA techniques represent the best available method to quantify protein carbonyl concentration” and “the inclusion of external standards allows standardization, better comparison, as well as high-throughput.”

BioCell Protein Carbonyl Assay Kit

10% discount when you buy 10 or more BioCell Protein Carbonyl Assay Kits.

That’s a saving of at least US$440.00. Other quotes available on request.

PROTEIN CARBONYLS

Protein carbonyls are biomarkers of protein oxidation that are generated by several different mechanisms during oxidative stress. They can be formed on arginine, proline, threonine and lysine residues as a result of metal-catalyzed oxidation. Protein carbonyls can also arise from the direct reaction of proteins with reactive oxygen species (ROS), or can be introduced into proteins by covalent attachment of carbonyl-containing molecules such as reducing sugars or lipid peroxidation products.

Key Benefits of BioCell Protein Carbonyl Assay:

  • Measures carbonyls quantitatively
  • Requires only microgram amounts of protein
  • Has a very low non-specific background
  • Is highly sensitive for analyzing clinical samples
  • Correlates well with the colorimetric assay
  • Easier to use and less labor intensive
  • Can handle more samples per day than colorimetic assays

Assay Principle:

Protein samples are reacted with dinitrophenylhydrazine (DNP) and then non-specifically adsorbed to an ELISA plate. Free DNP and non-protein constituents are easily washed away, meaning minimal interference.

The adsorbed protein is reacted with a biotinylated anti-DNP antibody followed by streptavidin-biotinylated horseradish peroxidase. Absorbance values are related to a standard curve prepared from serum albumin containing increasing proportions of HOCl-oxidized protein.

Using the kit:

  • Up to 89 samples can be analyzed without replicates, although duplicate or triplicate analysis is recommended for best results.
  • Carbonyl concentrations are calibrated against a protein that has been oxidized and standardized colorimetrically.

Stability:

BioCell Protein Carbonyl Assay Kits do not need to be kept under cool conditions for short-term transportation. The kits are stable at room temperature for up to six weeks. If the kit is separated into freezer, fridge and bench as per instructions on the leaflet, it is stable until at least the expiry date.

Background:

An ELISA test, using an anti-DNP antibody, was developed by: I. Hendrikje Buss, Christine C. Winterbourn and coworkers at the Centre for Free Radical Research, University of Otago, Christchurch, New Zealand

Frequently Asked Questions:

What samples can be analyzed?

You can use the protein carbonyl kit for any sample containing soluble protein. Biological samples that have been used include plasma, serum, urine, cell culture supernatant, cell extracts, tissue homogenates, bacterial and plant extracts, and oxidant-treated pure protein.

How stable are protein carbonyls?

Carbonyls are stable at -80 °C for months. However, levels change over that time in samples stored at -20 °C.

How does the kit perform?

Most healthy adult plasma protein carbonyls are around 0.05 nmol/mg. These values are elevated in many pathologies including diabetes, sepsis, chronic renal failure, acute pancreatitis, arthritis and coronary heart disease.

What should I expect for the Standard Curve?

Expect a linear curve with R-squared >0.95. A common reason for a non-linear standard curve is the substrate has been left too long before adding the stop solution and the top standards have saturated. Often the curve still fits very well when the highest standard is removed, or if sample values are also high, use a second order fit.

When first running the ELISA, it is a good idea to read the samples at 650 nm and save the results before adding the acid stop solution. It is recommended to add the solution when the highest standard/sample has reached an absorbance reading of 0.25 to 0.5.

Why do I get a poor concentration dependence with my standards?

This is usually because the freeze-dried standards have not been fully dissolved. With the current formulation they should dissolve readily, but if you are having problems, vortex vigorously at intervals over an hour at room temperature.

If my plate reader does not have a 450 nm filter can I read at another wavelength?

You can read within the 420-480 nm absorbance range.

How should ELISA plates be handled?

In reply to concerns about proteins sticking to the ELISA plate, the kit uses 3590 High Binding Plates, which the manufacturers guarantee have surface chemistry such that protein sticks and stays attached. We have washed hundreds (if not thousands) of plates extremely vigorously and never had a problem. However, do not to let the plates completely dry out at any stage.

How do I store the kit for maximum stability?

The kit is stable at room temperature only for 6 weeks after which its performance will decline. Only if the kit is separated into freezer, fridge and bench as per instructions, it is stable until the expiry.

The DNP solution should not be stored frozen or at 4 °C. DNP is in 6 M guanidine and is stable for many months if stored at room temperature in the dark.

The EIA buffer and blocking reagent can be stored frozen, and are stable for many months.

One user accidently froze the HRP-streptavidin. We tested this and found that it did not affect its performance.

What concentration are the standards and control and how long can the reconstituted standard stay at -80ºC?

Standards are at 40 mg/mL when reconstituted and remain stable at -80 °C for months or even years. Reconstituted standards and quality controls can be frozen and thawed twice.

How much protein is required to coat each well?

A key feature of the assay is that sufficient protein is added to saturate binding to the well. Under the conditions of the assay, approximately 1 μg of derivatized protein is added to each well. This is well over saturation and as a result, small variations in protein concentration can be tolerated.

If my absorbance readings are over range, can I dilute the samples?

No. Protein binding must be saturated in the assay. Halving the concentration should make no difference, and larger dilutions will give variable binding and unreliable results. The best way of overcoming high readings is to have a shorter color development time.

What is best for tissue extraction?

We do not recommend any particular method of tissue preparation. However, simple physical disruption works well. Tissue slicing, freeze thawing and homogenisation do not cause problems. Avoid detergents that could interfere with the DNP-derivatized protein coating the plate. We have also observed that some protease inhibitor preparations interfere due to high carbonyl content.
Extraction method: Wash/rinse tissue and homogenize vigorously in ice cold homogenisation buffer. We normally use 20 mM phosphate buffer (pH 7.4) containing 20 μM butylated hydroxytoluene (from stock dissolved in ethanol) and 100 μM diethylenetriamine pentaacetic acid to minimize oxidation during processing. Others have used 0.1% deoxycholate.
Aliquots of homogenate can be frozen at -80 °C and any precipitate should be centrifuged for 5 minutes at 10 000 rpm before assaying for total protein and carbonyl analysis.
For kit analysis, take an aliquot from each sample containing 20 μg of protein (all in an equivalent volume), add 0.8 volumes of 28% ice cold TCA and incubate for 10 minutes on ice.
Centrifuge at 10 000 g for 3 minutes (line lids up with outer edge of centrifuge) and aspirate from inner side of tube without disturbing pellet.
Add 5 μL of PBS and 15 μL of DNP to each sample and vortex.
The same DNP volume to protein volume/concentration must be maintained for samples and standards during derivatization. Therefore, when using extracts, the standards supplied in the kit must be diluted 1:10 to 4 mg/mL.
Take 5 μL of each diluted standard, add 15 μL of DNP and vortex.
Leave all samples at room temperature for 45 minutes to derivatize.
Take 5 μL, add 1 mL of PBS and vortex.
Proceed with usual kit instructions.

Publications using PC ELISA

Download PDF of publications: Publications Using Protein Carbonyl Assay Kit

Start typing and press Enter to search