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.

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.

Key Benefits:

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

Assay Principle:

Protein samples are reacted with DNP 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. Absorbances are related to a standard curve prepared from serum albumin containing increasing proportions of HOCl-oxidized protein.

Using the kit:

  • Up to 81 samples can be analysed in single analyses, 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 Free Radical Research Group, Christchurch School of Medicine (University of Otago), Christchurch, New Zealand.

Frequently Asked Questions:

What samples can be analysed?

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, oxidant-treated pure protein.

How stable are protein carbonyls?

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

How does the kit perform?

Normal range in human plasma is 0-0.150 nmol/mg. Most healthy adult plasma protein carbonyls are <0.1nmol/mg. Fo example in one study of 82 samples from 'healthy controls', the range was 0-0.20 nmol/mg, mean 0.025 (sd 0.045), & median 0 nmol/mg. Quality control - expected result 0.41±02 nmol/mg Intra-assay variance =2.6% Inter-assay variance= 5.5% Detection 0.008nmol/mg Previously <0.05nmol/mg usually around 0.02.

What should I expect for the Standard Curve?

Expect a linear curve with R 2 >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.

Why is my quality control value outside the range?

This is also likely to be a problem of solubility. The quality control may be more difficult to dissolve than the standards and is more likely to require the vortexing over an hour as described above.

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 4ºC. DNP is in 6M 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 & how long can the reconstituted standard stay at -80ºC?

Standards are at 40 mg/ml when reconstituted and remain stable at -80C for months or even years. Reconstituted standards & quality controls can be frozen & 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 conditions of the assay, 2 µg derivatised protein is added to each well. This is well over saturation and the same result is obtained with 1-3 µg. Variations in protein concentration within this range can therefore be tolerated. Standards should always be at the same protein concentration as samples.

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 colour 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 don’t cause problems. Avoid detergents that could interfere with the DNP-derivatised 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 homogenise vigorously in ice cold homogenisation buffer. We normally use 20 mM phosphate buffer pH 7.4 containing 20µM butylated hydroxytoluene (from stock dissolved in ethanol) and 100µM diethylenetriamine penta-acetic acid to minimize oxidation during processing). Others have used 0.1% deoxycholate

Aliquots of homogenate can be frozen at -80C and any precipitate should be spun down (5min @ 10,000rpm) before assaying for total protein and carbonyl analysis.

For kit analysis, take an aliquot from each sample containing 20μg protein (all in an equivalent volume), add 0.8 volumes of 28% ice cold TCA and incubate for 10min on ice.

Centrifuge at 10,000g/3min (line lids up with outer edge of centrifuge) and aspirate from inner side of tube without disturbing pellet.

Add 5μl PBS + 15μl DNP to each sample – vortex.

The same DNP volume to protein volume/concentration must be maintained for samples and standards during derivatisation. Therefore, when using extracts, the standards supplied in the kit must be diluted 1:10 to 4mg/ml.

Take 5μl of each diluted standard, add 15μl DNP and vortex.

Leave all samples at room temperature for 45min to derivatise.

Take 5μl, add 1ml PBS and vortex

Proceed with usual kit instructions

Selected Publications using PC ELISA

Buss H, Chan TP, Sluis KB, Domigan NM, Winterbourn CC.Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med. 1997;23(3):361-6.

Abu-Zidan FM, Winterbourn CC, Bonham MJ, Simovic MO, Buss H, Windsor JA. Small bowel ischaemia-reperfusion increases plasma concentrations of oxidised proteins in rats. Eur J Surg. 1999 Apr;165(4):383-9.

Winterbourn CC, Buss IH. Protein carbonyl measurement by enzyme-linked immunosorbent assay. Methods Enzymol. 1999;300:106-11.

Buss IH, Winterbourn CC. Protein carbonyl measurement by ELISA. Methods Mol Biol. 2002;186:123-8

Winterbourn CC, Chan T, Buss IH, Inder TE, Mogridge N, Darlow BA. Protein carbonyls and lipid peroxidation products as oxidation markers in preterm infant plasma: associations with chronic lung disease and retinopathy and effects of selenium supplementation. Pediatr Res. 2000;48:84-90.

Buss IH, Darlow BA, Winterbourn CC. Elevated protein carbonyls and lipid peroxidation products correlating with myeloperoxidase in tracheal aspirates from premature infants. Pediatr Res. 2000; 47:640-5.

Winterbourn CC, Buss IH, Chan TP, Plank LD, Clark MA, Windsor JA. Protein carbonyl measurements show evidence of early oxidative stress in critically ill patients. Crit Care Med. 2000;28:143-9.

Inder T, Mocatta T, Darlow B, Spencer C, Volpe JJ, Winterbourn C. Elevated free radical products in the cerebrospinal fluid of VLBW infants with cerebral white matter injury. Pediatr Res. 2002; 2:213-8.

Mocatta TJ, Winterbourn CC, Inder TE, Darlow BA. The effect of gestational age and labour on markers of lipid and protein oxidation in cord plasma. Free Radic Res. 2004; 38:185-91

Caimi G1, Hopps E, Noto D, Canino B, Montana M, Lucido D, Lo Presti R, Averna MR. Protein oxidation in a group of subjects with metabolic syndrome. Diabetes Metab Syndr. 2013:38-41

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