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General questions
Precautions and limitations
Sample preparation
Successfully running assays
Experimental data
To get the best results with our assay kits, we recommend reading the protocol book and the Precautions and Limitations, and Successfully Running Assays sections, before you start to prepare samples or reagents. For metabolite or enzyme activity assay kits, please also read the Sample Preparation and Experimental Data sections.
Please contact our Scientific Support team if you have any questions or need help after checking this article.
Is colorimetric or fluorometric detection more sensitive for microplate reader-based assays?
In general, fluorometric detection is about ten times more sensitive than colorimetric (spectrophotometric) detection. Check you have the right filters before buying a fluorometric kit.
Do you provide test samples?
Unfortunately, we do not offer test samples. Assay kits are included in our AbTrial program, for use in untested species or sample types without the financial risk.
At what temperature should I store the kit? What is the expiry date?
Storage temperatures are listed in the kit protocol book. If stored as recommended, assay kits can be stored for up to a year, unless the protocol book says otherwise. Reconstituted components can typically only be stored for two – three months.
Can you disclose the composition of the buffers in your kits?
Unfortunately, we cannot disclose assay buffer constituents or concentrations as they are proprietary information. A SDS (Safety Data Sheet) on the components can be found on each product datasheet with information on any hazards.
Does Abcam sell kit components separately? Can we use an alternate buffer for sample preparation?
In general, Abcam does not sell separate kit components, but some popular extra components are available. We highly recommend only 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.
What is your definition of a “test”?
Some kits are sold based on the number of “tests”. A "test" 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.
In which species or samples will the kit work (cells, tissue, serum, blood, plasma, etc ...)?
Our kits have been tested in human cells cultures/ lysates (unless stated otherwise), and most kits have also been tested in mouse and rat samples. Kits that detect generic metabolites (ammonia, carbohydrates, etc.) are expected to work in all species and their use is covered by the 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 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. If you would like to try our kits in a non-tested species or sample, you can benefit from our AbTrial program.
What are your recommendations for assaying multiple samples?
We recommend buying the same lot number when comparing or analyzing multiple samples. A new standard curve must always be prepared with each individual kit unit, however if ordering multiple kits from the same batch at the same time, we suggest comparing the standard curve of 2 kits, and if curves are identical, the same curve can then be used for other units of same batch.
Are kits ok to use when shipped over the weekend?
We recommend storing the kits at suggested temperature immediately after receiving. The shipping conditions over the weekend shouldn’t have any effect on quality.
What safety precautions should I take when using or handling the kit?
All reagents should be handled with care and disposed of properly. Please review the Safety Datasheet (SDS) provided for information. Always observe good laboratory practice; including use of gloves, lab coat, and protective eyewear, not eating, drinking or smoking in laboratory areas. All biological materials should be handled as if they are potentially hazardous and disposed of following established local safety procedures.
Can I use your products for diagnostic procedures?
No. Our products are intended for research use only.
Are there any other materials required to use your kits which are not listed in the protocol?
Our kits require materials and tools commonly found in research laboratories, including: MilliQ water or other type of double distilled water (ddH2O), pipettes and pipette tips including multi-channel pipettes, assorted glassware for the preparation of reagents and buffer solutions, and tubes for the preparation of reagents and buffer solutions.
Why do I have some leftover reagents after performing the assay?
Selected components in our kits are supplied with surplus amounts to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures.
Can I modify the protocol, or mix or substitute reagents or materials from other kit lots or vendors, and still expect good results?
The components of each kit have been carefully formulated and tested together, on a lot-specific basis, using the protocol provided. We understand that, occasionally, you might want to modify the protocol, to extend the use of the kit by adding in separate components from other kits, or to use individual components in other assays. We cannot guarantee performance in these circumstances.
Should I deproteinate my samples when using an enzymatic assay to measure a metabolite? Which method should I use?
Detection of many metabolites such as NADH, NADPH or ATP can be hampered by the 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 using trichloroacetic acid (TCA) to precipitate proteins, followed by a neutralization step. This ensures precipitation of interfering proteins with minimal loss of metabolites.
Perchloric acid (PCA) is also commonly used for sample deproteinization, however, it is more dangerous and corrosive than TCA. 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: metabolites are collected in the flow-through while enzymes/proteins are trapped in the filter. We recommend 10kD spin columns. 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 block the filter.
Can I freeze and store my samples prior to analysis with enzyme activity and metabolite assays?
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 analyte of interest we suggest deproteinization (typically required for metabolite assays), followed by snap freezing in liquid nitrogen, and storage at -80°C until use. Samples should undergo minimal freeze-thaw cycles (aliquot samples before freezing to avoid multiple freeze-thaws) 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.
How should tissues be prepared for analysis with enzyme activity and metabolite assays?
Tissue 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 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 lysate preparation guide for a general preparation procedure.
Failure to consistently and completely homogenize tissues can lead to erratic readings.
Some substances can interfere with assays and should be avoided in sample preparation. In general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) interfere with many enzyme-based assays and should be avoided in sample preparation.
I don't have enough cells for your recommended starting amount. Can I scale down the buffers?
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 (this is not appropriate for assays to be run on live or fixed cells).
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount of any sample to be used since it is sample concentration and quality based. We recommend you run a pilot experiment with multiple dilutions to determine the optimal dilution which gives a reading within the linear range of the standard curve.
What tips can you give for running assays?
We recommend that for all assays you consider the following to ensure that you get the best results:
Preparing reagents
- avoid foaming or bubbles when mixing or reconstituting components.
- avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.
- ensure plates are properly sealed or covered during incubation steps.
- ensure all reagents and solutions are at the appropriate temperature for use before starting the assay (i.e. not taken straight from the ice bucket into the assay) and mix reagents gently before use.
- don’t allow reagents to sit for extended periods before use, or reuse prepared reagents, always prepare fresh reaction mix before use.
- make sure that all equipment is switched on and set at the appropriate temperature before starting the assay.
Performing the assay
- ensure that you accurately follow the recommended incubation times and temperatures in the protocol.
- when preparing both reagents and samples and performing the assay, avoid pipetting small volumes (< 5 µL) and prepare a master mix where possible; this will reduce variability in results.
- avoid air bubbles in wells, which can distort readings, by pipetting gently against the wall of the tubes.
Reading the assay
- carefully check that you are using the correct wavelength and filter settings for the assay.
- ensure that you are using the right plate type:
Colorimetric: clear plates
Fluorometric: black wells/clear bottom plates
Luminometric: white wells/clear bottom plates
Assay analysis
- interfering substances in samples can distort results, read the protocol carefully to identify these and also deproteinize samples where recommended.
- for assays with a standard curve, with samples with analyte concentrations greater than the most concentrated standard, to obtain an accurate reading you should dilute the sample in the appropriate sample dilution buffer to bring it within the linear range of the standard curve.
For assays with a standard curve, how do I calculate my values?
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 a 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.
Why are my standard curve values lower than those shown on the datasheet?
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.
Why is sample normalization necessary?
When comparing the enzymatic activity of 2 or more samples or analyzing the effect of inhibitors or agonists on enzymatic activity between samples, it is always necessary to normalize the samples based on the number of cell or the protein concentration after cell lysis.
What are expected OD readings using sample X?
If we know of expected values using certain sample types, we will add this information to our datasheet.
How long do assays take?
The protocol times vary depending on the kit, analyte, and whether the samples require additional purification steps prior to measurement. If we are 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.
What is the range of sensitivity of the assays?
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.
My microplate reader does not have the filter recommended on the datasheet. Can I still use the assay?
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.