Immunoassay Kit Specific FAQs

Q: When is it appropriate to use the Assay Designs Direct, Regular, or Complete cyclic AMP or cyclic GMP EIA kit?
A: The Direct kits are best used for cell lysates and tissue homogenates (e.g. intracellular cyclic nucleotide). The kit includes 0.1M HCl which is used to lyse or homogenize the samples. The Regular kits are used when measuring cyclic nucleotides in biological fluids, culture supernatants, or samples that require extraction and concentration (e.g. extracellular cyclic nucleotide). The Complete kit combines the buffers supplied in both the Direct and Regular kit to allow you to measure either intra- or extracellular cyclic nucleotide with one convenient product.

Q: How do I know if I should acetylate my samples?
A: You should acetylate your samples if they contain a very low concentration of cAMP or cGMP, or when greater sensitivity is required. The sensitivity with acetylation is approximately ten-fold greater than the non-acetylated format. If you have no idea what the levels of cyclic nucleotide will be in your samples, we recommend trying a few non-precious samples without acetylation first in order to determine if this step is necessary for you.

Q: Can I store acetylated samples for analysis at a later time?
A: No. We recommend that you acetylate only enough sample as is needed for the current run. Samples will be more stable if stored without acetylation reagents added to them.

Q: How do I treat my cells with the 0.1M HCl provided with the Direct and Complete cAMP/GMP kits?
A: The HCl is added to lyse your cells, stabilize the cAMP/cGMP, and inactivate intracellular phosphodiesterase activity. It is extremely important to thoroughly aspirate off all media or buffer prior to the addition of HCl. Add just enough 0.1M HCl to cover the cells, shake gently for 10 minutes and visually inspect cells to verify cell lysis. If adequate lysis has not occurred, incubate for a further 10 minutes and inspect.

Q: What if my cells have still not lysed after the addition of 0.1M HCl?
A: Cell or tissue lysis can be enhanced by adding 0.1% to 1% Triton X-100 to 0.1M HCl prior to use. When used in this concentration range, the detergent will not interfere with acetylation or the binding portion of the assay; however there will be a modest increase in the optical density. Samples containing Triton X-100 should be evaluated against a standard curve diluted in the same for the most accurate determination.

Q: I am not getting consistent results with my detection in cells. I think that my cells may be permeabilized.
A: For the most consistent results, we recommend that cells be fully disrupted before using the lysate in an assay. Disruption should be verified by microscopic examination since cell lines vary in their hardiness.

Q: Why is a small volume of concentrated HCl suggested for lysing liquid samples?
A: Cyclic nucleotides are normally found in low concentrations. Using concentrated acid minimizes sample dilution.

Q: Is it necessary to use glass tubes for the cyclic nucleotide assays?
A: We recommend glass tubes when acetylating samples as the acetylation reagents tend to melt polystyrene. Glass or polypropylene tubes should be used to avoid this problem.

Q: How should I store my cell lysates prior to analysis?
A: Cyclic nucleotides will be very stable in the 0.1 M HCl used for lysis and the low pH will inhibit further synthesis and metabolism. It is generally suggested to pellet the precipitate and store the supernatant at -80°C.

Q: How much cyclic AMP/GMP is there per cell? How many cells should I start with?
A: The amount of cyclic nucleotide per cell will vary by cell type, volume, and passage. We recommend that you begin with a large number of cells, and make dilutions of the sample to determine the appropriate sample conditions for your specific application. Remember, it is easier to dilute a concentrated stock than to start your experiment all over again.  

Q: My maximum binding for acetylated standards and samples is only about 50%. Why?
A: This occurs if the standards and samples were acetylated but the Bo standard was not. Your data can still be used, simply calculate sample concentrations off the optical density curve and do not perform the %B/Bo analysis.

Q: What antibody pairs are used in the kit?
A: This varies from kit to kit. Refer to the product specific instruction manual for more information

Q: Will the cytokine kits from one species work with another species?
A: This will depend on the species homology. Human cytokine kits generally will NOT measure cytokines in other species. Mouse or rat cytokine kits will NOT measure human cytokines unless otherwise specified.

Q: Do eicosanoid assays require an extraction?
A: The majority of samples do not require an extraction. However, in some situations an extraction will be required when the expected levels are below the detection range of the kit or the sample contains interfering substances.

Q: What is the purpose of a Urinary Prostacyclin kit if I can use your 6-keto-PGF_1α kit for the measurement of prostacyclin?
A: In urine, prostacyclin is broken down into 6-keto-PGF_1α, which is subsequently broken down into 2,3-dinor-6-keto-PGF_1α. Thus, to obtain a true estimate of prostacyclin in urine, both 6-keto-PGF_1α and 2,3-dinor-6-ketoPGF_1α should be measured. The Urinary Prostacylin kit will allow for both of these measurements. The breakdown of prostacyclin is not the same in other matrices.

Q: How much indomethacin should I use in my samples?
A: You should follow the suggestion in the product specific manual, and add up to 10 ug per ml of liquid sample. The synthetase inhibitor will ensure that the synthetic cascade is shut down, thereby avoiding changes in the sample concentration post-collection.

Q: What species can the eicosanoid kits be used for?
A: Eicosanoids are not species-specific. However, the antisera used in a particular assay may present an interference effect due to the species of IgG used on the plate. In this case, the kit can still be used but the endogenous IgG will need to be removed. This can be done by precipitating out the sample IgG prior to running the assay or by extracting the samples with the extraction protocol suggested in the kit insert.

Q: What is the difference between the 8-iso-PGF_2α kit and the Direct 8-iso-PGF_2α Kit?
A: The regular 8-iso-PGF_2α kit is designed to measure free isoprostane in samples. Direct 8-iso-PGF_2α measures total isoprostane levels, both free and esterified. This is accomplished by hydrolyzing the ester bond prior to analysis.

Q: I am using the Direct 8-iso PGF_2α kit. After I add the HCl to neutralize my sample it is not reading in the range of pH 6-8. What should I do?
A: At this point you have added HCl to NaOH which will yield NaCl + H₂O. There is little buffering capacity allowing the pH to fluctuate quite easily. Simply bring the pH to the range of 6-8 and continue on with the procedure.

Q: If I have 2N NaOH hydrolyzed samples for an 8-iso-PGF_2αdetermination, do I need to extract these samples?
A: No, the Direct 8-iso-PGF_2α kit is specifically designed to help you obtain results as quickly and easily as possible. The hydrolyzed samples can be measured in the assay by simply following the directions outlined in the kit insert.

Q: Do I need to add Steroid Displacement Reagent to my samples in Assay Designs steroid kits?
A: If total steroid levels are to be measured, Steroid Displacement Reagent (SDR) should be added to the samples containing binding proteins before running in the assay. The reagent will preferentially associate with steroid binding proteins to the exclusion of the steroid itself. Once free of the binding proteins, the steroid can be measured in the kit. Samples that are extracted do not require the addition of SDR.

Q: Can I measure urinary steroid levels in Assay Designs kits?
A: Steroids are relatively insoluble and must be altered by being conjugated to glucoronic acid for elimination. The antibodies in our kits will not recognize conjugated urinary steroids. If desired, chemical treatment can be employed to break the glucuronide bond.

Q: In the sPLA2 (Cat. # product.php?pid=ADI-907-002ADI-907-002) activity kit, how is a Unit defined?
A: One unit of phospholipase activity is the amount of enzyme needed to hydrolyze 1.0 μmole of L-α-phosphatidylcholine to L-α-lysophosphatidylcholine and a fatty acid per minute at the appropriate pH and temperature.

Q: What type of media is best for use in the H₂O₂ assays (Cat. #ADI-907-012 and ADI-907-015)?
A: Media containing phenol red as a pH indicator will interfere in the assay. Most media are available with this removed as an option. DMEM should not be used in the colorimetric assay due to the ferrous sulfate salts that it contains. Serum cannot be added to the media for the chemiluminescent assay because hemoglobin is used as the trigger.

Q: How do I know whether to use the Regular or Total Nitric Oxide kit?
A: The regular kit measures both major metabolic products of NO -- nitrate and nitrite. The Total kit measures these metabolites as a single quantity after the sample has been treated with Nitrate Reductase. Kit selection will need to be determined by the needs of the individual researcher.

Q: Can I measure tissue NO in the Nitric Oxide kits?
A: Yes. Samples can be homogenized in the provided Reaction Buffer and then centrifuged. This supernatant should then be filtered through a 10,000 MWCO device, diluted (if necessary) and run in the assay.

Q: Can I run cell lysates in the Nitric Oxide kits?
A: Yes. First remove the media from the cells. Pellet them and wash with PBS before lysing the cells in the Reaction Buffer provided in the kit.

Q: My cell culture samples are in DMEM. Will this interfere in the Nitric Oxide assay?
A: Yes. The nitrate salt used in this particular media will cause interference. It is important to check the published media formulation to ensure that excessive levels of nitrates are not present.

Q: Why do I have to filter my samples through a 10,000 MWCO filter?
A: In order to get the best sensitivity, samples need to be filtered to remove or reduce interfering substances. Using a 10,000 MWCO, many proteins and larger complexes will be eliminated while nitrate and nitrite will easily pass through.

Q: What type of 10,000 MWCO filter is best to use?
A: The user has a choice between a spin-filter, a syringe-type filter or 96-well filtration plate formats. Generally a filter made of polysulfone is recommended. These can be obtained from most general lab supply providers. Often, plasma and serum will be difficult to filter due to the presence of proteins and lipids. If a dilution is required, this can be done in part prior to filtering to make this process easier.

Q: It says in the insert that samples containing high levels of NADPH need to be diluted sufficiently. How can I determine the NADPH levels and dilution?
A: We have found that 0.5 - 1 mM NADPH will inhibit the Griess color reaction slightly. If this amount of NADPH is present the sample ODs can be artificially but modestly depressed. In order to determine the dilution necessary, a serial dilution of a spiked sample can be run. If there is no interference, the recoveries will be linear.

Q: I am seeing much higher than expected nitrate levels. What could be the problem?
A: Many times the water source in the lab is the culprit. The water is only as pure as the last filter that it passes through. It is important that these filters be checked regularly. To check if this is the problem, you can simply run water as a sample in the assay.

Q: Can I use the COX activity kit to distinguish between COX-1 and -2?
A: The COX Activity kits measure activity alone, distinguishing between COX-1 or -2. In order to differentiate between the two enzymes, two COX inhibitors are included in each kit. Ibuprofen can be used as a non-specific inhibitor while NS-398 is COX-2 specific. The use of these two compounds will allow the researcher to determine how much activity is due to each COX enzyme in a given sample.

Q: Can I use cell culture samples in the COX Activity assay?
A: Cell culture samples are compatible with this kit. Since this is an activity assay rather than a quantitative immunoassay, there is no extraction necessary. The Tris-phenol buffer described in the kit insert is suitable for lysing cells using physical methods. If a different buffer is used for lysis, the end-user will have to test the buffer for compatibility in the assay.

Q: Do I need a luminometer with double injectors for this assay?
A: Yes. A single injector will not provide good results. You cannot pre-mix the arachidonic acid and substrate to make a single addition trigger, nor can you add either one by hand and allow the instrument to add the other. This instrument configuration is required for accurate readings.