Q: Is there an advantage to using the Protein Carbonyl ELISA Kit compared to the fluorometric or spectrophotometric formats?
A: Our Protein Carbonyl ELISA kit is about 5-10 times more sensitive than our Protein Carbonyl Spectrophotometric Assay and only requires 1 µg of protein per assay versus 250 µg protein per assay with the spectrophotometric format. Many more samples can be processed at once in the ELISA kit, while the spectrophotometric kit is performed in individual tubes. The spectrophotometric kit format is designed for use by laboratories without an ELISA plate reader.
Q: How should I prepare my samples?
A: The protein carbonyl ELISA requires all protein samples to be diluted to 10 µg/mL, which are used to coat the plate. If preparing cell or tissue lysates, detergents such as Triton X-100 or NP-40 should be avoided as they can interfere with protein coating. We recommend resuspending cells or tissues in PBS containing proteinase inhibitors and optional 0.005% Butylated hydroxytoluene (a generic antioxidant available from Sigma-Aldrich; you can prepare 5% stock of BHT in methanol), followed by either homogenization or sonication, then centrifuge at 12000 xg for 10 min and harvest the supernatant as your lysate. The protein concentration should be determined with a protein assay, such as BCA or Bradford, and samples can then be diluted to 10 µg/mL in 1X PBS and used to coat the plate.
Q: Can I increase the amount of protein for coating to increase the signal?
A: Coating with more protein will not increase the signal. All wells are coated with 100 µL of 10 µg/mL protein, which is 1 µg protein per well. This concentration is in excess of the protein binding capacity of the well to ensure the maximum amount of protein carbonyl is captured. Adding more than this will not result in capturing more protein carbonyl or an increased signal.
Q: Can I vary the amount of protein in the samples?
A: All samples must be at 10 µg/mL because this is an indirect ELISA where the plate is coated with the samples as antigens, rather than with an antibody. It is important that all samples are at the same concentration. You will first measure the protein concentration of your samples using a protein assay such as BCA or Bradford, and then dilute samples to 10 µg/mL in 1X PBS.
Q: I have several samples; do I have to determine the protein concentration for all of them?
A: Since it isn’t practical to measure the protein concentration of all samples, you can measure a few of the samples and then use the average as an estimate for all the samples.
Q: Can I prepare blood samples using heparin or EDTA?
A: Our Protein Carbonyl ELISA can be used for any protein samples containing protein carbonyl modifications whether it’s lysate, serum or plasma. To harvest plasma, you can use either Heparin or EDTA; neither will interfere with the assay.
Q: What is considered a high nucleic acid concentration that will interfere with the assay?
A: For most samples, the nucleic acids do not need to be removed; it is only required if you are using whole cell lysate. Nucleic acids contain keto groups with can react with the DNPH reagent, producing a false signal. If you are unsure if this step is necessary, a representative sample can be prepared with and without the precipitation to determine if it is necessary. To remove nucleic acid, add streptomycin sulfate or PEI to a final concentration of 1% or 0.5% respectively, incubate 30 minutes at room temperature and remove the nucleic acid precipitates by centrifuging at 6000 xg for 10 minutes at 4ºC.
Q: Is it possible to replace PEI and streptomycin sulfate with benzonase to remove nucleic acids?
A: Any DNA/RNA removal method can be used as long as no additional proteins are introduced during the procedure. Any additional proteins may contain carbonyl groups, which will influence the assay results.
Q: Is this assay species specific?
A: The structure of protein carbonyl groups is the same regardless of species; therefore any protein sample from any species can be used with this assay.
Q: Which is better, incubation at 37ºC for 2 hours or 4ºC overnight?
A: Long and slow incubations are always preferred, but it is not essential. You will still get good results with a two hour incubation at 37ºC. The incubation time should not affect variability within the assay as long as you are consistent with the time you select.
Q: How do I know when to add the Stop Solution?
A: A large incubation range is provided because the development time can vary, which is typical for any ELISA. Upon adding the TMB substrate, a blue color will slowly develop, which will then change to yellow after adding the stop solution. If samples are incubated too long in substrate solution they can become saturated. To determine when to add stop solution, focus on the wells with the standard curve where you will want to see a bright blue color develop for the highest concentration and a very faint color in the lowest concentration. When you see a nice gradient of color you should add the stop solution and read the plate immediately. It is important to add the stop solution to all the wells at the same time, using a multichannel pipettor, regardless of the difference in colors between samples.
Q: How do I make the standard curve and calculate my results?
A: The best way to determine concentrations from a standard curve is to use a 4-parameter curve fitting program, but if you don’t have this software it can be done using Excel. Graph the standard curve and generate a linear trendline. The standard curves generated with ELISAs are not typically linear, but a linear curve can be created by eliminating the upper and lower values of the curve, as long as the sample values fall within this range. The middle part of the standard curve is the most sensitive and is the best part to use for quantifying samples. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is (OD value-b)/m for linear lines.
Q: Why are my standard curve ODs so high even at low concentrations?
A: The high signal and fast development time may be a result of excess DNPH that was not removed during the wash steps. DNPH is a very sticky molecule, therefore the washing in step 5 is very critical to remove any free DNPH: "Wash wells with 250uL of 1X PBS/Ethanol (1:1, v/v) with incubation on an orbital shaker for 5 minutes. Repeat washing a total of 5 times, aspirating between each. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess wash solution. Wash 2 times with25 uL of 1X PBS."
To reduce the development time, we recommend repeating the assay with just the standard curve and with thorough washing. Another option to slow the reaction is to add less antibody in the ELISA. For example, instead of preparing a 1:1000 dilution of primary and secondary antibody, prepare at 1:2000.
Q: What should I do if my sample is running higher than my standard curve?
A: In this case you will need to further dilute your sample, but without diluting the protein concentration. You can accomplish this by diluting your 10 µg/mL protein sample in 10 µg/mL Reduced BSA that you prepared as part of the standard curve. You may need to try a couple of dilutions to find the one that falls within the standard curve range.