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What is the assay principle? What is the end product I am detecting?
We have two types of cellular and biochemical assay kits: enzymatic and antibody-based assay kits.
Which type of detection method is more sensitive?
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.
Do you provide small testing samples?
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 application or species without the financial risk.
At what temperature should I store the kit? For how long can I store it?
The proper storage temperature for your kit will be described in detail on the protocol booklet that we send you along with the kit. If some of the components are stored at a different temperature, it will be clearly indicated in the protocol booklet.
If stored upon receipt, our assay kits can generally be stored up to a year although if that is not the case it will be clearly stated on the protocol booklet. For kits where components need to be reconstituted, the shelf life of the components will change to two months after reconstitution.
Do you have any details on the buffer components of your kits?
Assay buffer compositions and concentration cannot be publicly disclosed as they are proprietary information. A MSDS (material Safety Data Sheet) on the components can be found on each product datasheet which contains information on hazardous components and hazard identification.
Does Abcam sell the kit components separately, in case I need additional buffer?
In general, Abcam does not sell separate kit components, but we have some popular extra components available for sale. If extra components are available for a specific kit, it will be indicated on the datasheet.
What is the definition of your test sample?
Our kits are sold based on number of tests. A "test" simply refers to a single assay well, including controls and standards necessary to perform the assay. The number of wells that contain sample, control or standard will vary depending on the product. We recommend reviewing the protocol completely to confirm it meets your requirements. Please contact our Scientific Support team if you have any questions.
Should I deproteinate my samples? Which method should I use?
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 degraded and the stability of the metabolites will be improved.
The most efficient protocol consists on precipitation of proteins using perchloric acid (PCA) followed by a neutralization step - please see our Deproteinization Protocol for a detailed step by step protocol.
Alternatively, spin colums can be used as a quicker alternative to the deproteinization step: the metabolites are collected in the flow-through while the degradating enzymes 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. The one thing to consider, though, is that the smaller the size of the filter, the harder it is to spin the column dry so that all the liquid is recovered. As a result, you may lose metabolite.
We would suggest following the PCA-based Deproteinization Protocol whenever possible as it ensures precipitation of interfering proteins with minimal loss of metabolites.
Can I use a sample prepared with your preparation kits in other assays (mass spectrometry, western blot, etc ...)?
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.
Can I freeze and store my samples prior use?
We recommend that you prepare your sample immediately prior to measurement to obtain the best results. However, we understand that this is not always possible due to experimental conditions. If you want to freeze your samples, we suggest deproteinization 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.
How should tissues be prepared for analysis?
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.
Some substances can interfere with the assays and should be avoided in sample preparation. Please see individual kit datasheets 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. Moreover, we recommend deproteinizing the sample prior to analysis using the Deproteinization protocol.
In which species or samples will the kit work (cells, tissue, serum, blood, plasma, etc ...)?
All of our kits have been tested in human cells in culture (unless stated otherwise), and most of them have been tested in mouse and rat as well. Due to the nature of the detection method in our enzymatic assays, we expect they will work with most mammalian species although this may require additional optimization. Any other information on the tested species will be stated on the datasheet.
The sample types in which the kit has been tested will be displayed in the product datasheet.
If the species or 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.
I don't have enough cells for your recommended starting amount. Can I scale down the buffers?
We have optimized each assay with 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 on the protocol.
If that is not possible, we would recommend harvesting cells at different times and freeze them till there is enough sample to proceed wih the protocol.
How do I calculate my values from the standard curve?
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 Analysis of results from a standard curve.
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. You might also want to check the "Scientific Support" and "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.
How long do assays take?
The protocol times vary depending on the kit, analyte, and whether the samples require additional purification steps priot 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.
What is the range of sensitivity of the assays?
Each assay has different sensitivity, depending on what it s 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.
My microplate reader doesn't have the filter recommended on the datasheet. Can I still use the product?
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 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.