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In general, fluorometric detection is about ten times more sensitive than colorimetric (spectrophotometric) detection. Fluorometric detection usually requires specific filters for optimal excitation and emission of the fluorophore.
No, at the moment we do not offer testing samples. However, our assay kits are included on Abcam's AbTrial program, where you can use our products in an untested species or sample type without the financial risk. Contact our Scientific Support team to discuss other possibilities. All Abcam kits are guaranteed to work in tested species and sample types.
The proper storage temperature is described in detail on the protocol booklet that we send along with the kit. If some of the components required to be stored at different temperature, then for guidance please refer to the protocol booklet.
If stored as recommended, our assay kits can be stored up to a year, although if that is not the case it will be clearly stated on the protocol booklet. For the kits where components need to be reconstituted, the shelf-life changes to two – three months after reconstitution. Please contact our Scientific Support team if you have any questions.
Assay buffer compositions and concentration cannot be publicly disclosed as they are proprietary information. A SDS (Safety Data Sheet) on the components can be found on each product datasheet which contains information on hazardous components with hazard identification.
In general, Abcam do not sell separate kit components, but we have some popular extra components available for sale. Please contact our Scientific Support team if you are in need of any extra component.
Assay buffers are optimized for the reactions they are designed for. They not only contain detergents for efficient lysis of cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results. Do not mix components between different kits.
If a lysis buffer is not provided, please follow the protocol recommended in our tissue and cell preparation guide.
Our kits are sold based on number of tests. A "test" simply refers to a single assay well, and controls and standards necessary to perform the assay are also included in this number. The number of wells that contain sample, control or standard will vary depending on the kit. We recommend reviewing the protocol completely to confirm it meets your requirements.
Detection of many metabolites such as NADH, NADPH or ATP can be hampered by presence of interfering enzymes that would degrade the metabolite. By deproteinizing the sample, the interfering enzymes are removed and the stability of the metabolites improves.
For a quick and efficient deproteinization, we suggest using Deproteinizing Sample Preparation Kit – TCA (ab204708). This kit is based on the ability of trichloroacetic acid (TCA) to precipitate proteins, followed by a neutralization step. This process ensures precipitation of interfering proteins with minimal loss of metabolites.
Perchloric acid (PCA) is another common compound used for sample deproteinization, however, it is a more dangerous and corrosive acid than TCA and might not be available to many researchers. For an alternative step by step protocol using PCA, follow our PCA-based Deproteinization Protocol.
Spin columns can also be used as a quick deproteinization step: the metabolites are collected in the flow-through while the enzymes/proteins are trapped in the filter. We recommend 10kD spin columns, although it is possible to use smaller sized spin columns (such as 3kD) if the compound of interest has a small enough molecular weight to pass through the filter. Small sized filters are hard to spin and dry so these can result in loss of metabolite. We recommend using 10kD spin columns only for liquid samples, as cell and tissue lysates will have debris that can potentially block the filter.
Yes, you can use the sample obtained with our preparation kits in any assay for which the sample may be suitable. Just make sure that the sample preparation buffers are compatible with the downstream assay you want to perform.
We recommend using fresh samples wherever possible for best results. However, we understand that this is not always possible due to experimental conditions. Depending upon the metabolite of interest we suggest deproteinization (if required), followed by snap freezing in liquid nitrogen, and storage at -80°C until use. Samples should undergo minimal freeze-thaw cycles and never be stored for long periods of time. This is extremely important for metabolites with short half-life or for proteolytic enzymes that can lose activity after storage.
Samples should be completely homogenized on ice in cold sample preparation buffer, preferably using a Dounce homogenizer. The amount of buffer necessary should be indicated on the protocol; if that is not the case, use a volume between 4 - 6 times the tissue volume. Carefully transfer the homogenate to a tube (microcentrifuge, 15 mL or 50 mL tube depending on the amount of tissue) and centrifuge the sample at low speed in a cold centrifuge for 3 - 5 minutes. Transfer the supernatant to a new tube and discard the insoluble pellet. Read our tissue and cell preparation guide for a general preparation procedure.
Some substances can interfere with the assays and should be avoided in sample preparation. Please see individual kit protocols for more specific information on which substances may affect the specific kits, but in general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) are known to interfere with these assays and should be avoided in sample preparation.
All of our kits have been tested in human cells cultures/ lysates (unless stated otherwise), and most of them have also been tested in mouse and rat samples. Kits that detect generic metabolites (ammonia, urea, carbohydrates, hormones etc.) are expected to work in all species and their use is covered by Abpromise guarantee. Due to the nature of the detection method of our enzymatic activity kits, we expect they will work with most mammalian species and with other organisms such as bacteria, plants, drosophila or yeast; although this may require additional optimization. The sample types in which the kit has been tested are displayed on the product datasheet or protocol booklet.
If the sample you wish to analyze is not listed on the datasheet, please contact our Scientific Support team for more information. If you would like to try our kits in a non-tested species or sample, you can benefit from our AbTrial program.
We have optimized each assay for the cell number stated in the protocol. You can try to scale down, and decrease the amount of buffers used proportionally, but it may not fall within the range for the kit. Therefore, we encourage users to follow the sample recommendation in the protocol. If that is not possible, we would recommend harvesting cells at different times and freezing them until there is enough sample to proceed with the protocol.
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot experiment with multiple dilutions to determine the optimal dilution which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Typical experimental results will be determined by comparing the value of the sample to that of a standard whose value is known. You can find more details on how to calculate results back from the standard curve in the protocol booklet's Data Analysis section.
If you need help generating the standard curve or using it to determine the concentration or value of your metabolite of interest in each sample, please check our helpful resource guide on calculating and evaluating ELISA data.
There are multiple factors which can influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
If we know of expected values using certain sample types, we will add this information to our datasheet. You might also want to check the "specific references" sections of the datasheet for more information. If we don't have the information listed, you may want to consult the latest literature or contact our Scientific Support team.
The protocol times vary depending on the kit, analyte, and whether the samples require additional purification steps prior to measurement. If we were able to standardize the protocol time, this information will be included on the datasheet. However, be aware that this is only an indication, and you should plan your experiment carefully and after thoroughly reading the protocol.
Each assay has different sensitivities, depending on what it is measuring and the detection method used. We have added the information to the datasheet when available. If you have any queries regarding your sample and cannot find it on the datasheet, please contact our Scientific Support team.
The filter recommended on the datasheet will detect the absorbance and/or fluorescence emission of the dyes and reagents at its maximum and optimal wavelength, which is very important if you have low amount of sample or weak enzyme activity. Filters that differ from the optimal wavelength +/-20 nm could still be used; however, the further they are from the peak the weaker the signal will be, which can lead to higher background and noise.
If you are not sure whether your filter can be used to determine a specific wavelength, please contact our Scientific Support team who will be able to advise you on that matter.