FAQs
You can use the Protein Carbonyl Assay 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.
Carbonyls are stable at -80 °C for months. However, levels change over that time in samples stored at -20 °C.
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.
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.
You can read within the 420-480 nm absorbance range.
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.
The kit is stable at room temperature for 6 weeks, after which its performance will decline. Only if the kit is separated into freezer, fridge and bench as per instructions, is it stable until the expiry date.
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.
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 derivatised protein is added to each well. This is well over saturation and as a result, small variations in protein concentration can be tolerated.
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.
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 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 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 minimise 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 biotin hydrazide to each sample and vortex.
The same biotin hydrazide 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 4 mg/mL.
Take 5 μL of each diluted standard, add 15 μL of biotin hydrazide and vortex.
Leave all samples at room temperature for 45 minutes to derivatise.
Take 5 μL, add 1 mL of PBS and vortex.
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Frequently Asked Questions
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