- Why do some protocols recommend using 10kD spin columns (ab93349)? Should I deproteinate my samples?
- I have 3kD spin off filters. Can I use these instead of the 10kD?
- How do I calculate my values from the standard curve?
- What are expected OD readings using sample X?
- In which species has this been tested?
- Will this work with my samples (serum, blood, plasma, cell medium, etc ...)?
- How long do assays take?
- Can I freeze and store my samples prior to use?
- What is the assay principle? What is the end product I'm detecting?
- Which is more sensitive, the fluorometric or colorimetric assay?
- What is the range of sensitivity of the assays?
- How should tissue be prepared for the kits?
- Do you have any details on the component of buffers of your kits?
- Has the assay been performed with fewer than 1x106 cells? Do I scale down the buffers?
- I need more extraction buffer. Does Abcam sell this separately?
1. Why do some protocols recommend using 10kD spin columns (ab93349)? Should I deproteinate my samples?
Detection of many metabolites such as NADH or NADPH can be hampered by presence of interfering enzymes that would degrade the metabolite. By filtering the samples through the spin column, the metabolites are collected in the flow-through while the degradating enzymes are trapped in the filter, improving the stability of the sample.
It is possible to use a smaller sized spin column 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 recommend using the 10kD spin column (ab93349) for this reason.
Typical experimental results will be determined by comparing the value to that of a standard whose value is known. If you need help generating the standard curve or how to use it to determine the concentration or value of your metabolite of interest in each sample, please check our helpful resource guide 'Calculation of results from a standard curve'.
If we know of expected values using certain sample types, we will add this information to our datasheet. You might also check the "Scientific Support" and "Specific references" sections of the datasheet for more information. If we don't have the information listed, you may consult the latest literature or contact our Scientific Support team.
All our kits have been tested in human cells in culture, and most of them have been tested in mouse and rat, as well. Information on the tested species can be found on the datasheet. Due to the nature of the detection, we expect they will work with other mammalian species although it might require additional optimization. Contact our Scientific Support team for more information.
If you would like to try our kits on a non-tested species, you can benefit from our 100% Testing Discount scheme.
This information is available on the datasheet. If your sample is not listed or you have any queries regarding the suitability, please contact our Scientific Support team.
The experimentation times vary depending on the kit and what is trying to measure. We have added this information to the datasheets, but please be aware that it is only an indication; you should plan your experimental timings carefully and after thoroughly reading the protocol.
We recommend that you prepare your sample immediately prior to measurement to obtain the best results. This is, however, not always possible due to experimental conditions. If you want to freeze your samples, we suggest you deproteinate them first (for instance, using 10kD spin column (ab93349)) and then snap freeze the samples using liquid nitrogen followed by storage at -80 C until use. Samples should undergo minimal freeze-thaw cycles.
We have two types of assay kits: enzymatic and antibody-based assay kits.
Our enzymatic assay kits are based on detecting product formation through a specific enzymatic reaction. Each assay kit is specific and will quantify either the direct product or a specific metabolite whose formation is proportional to the product to detect.
We cannot disclose the exact working mechanism of our assays as this is proprietary information. If you have a query about a particular kit, please contact our Scientific Support team for more information.
Our antibody-based assays are ELISA-like assays, where an antibody detects the final product, whether it is a metabolite or a protein.
Fluorometric detection is generally more sensitive than colorimetric (spectrophometric) detection. Fluorometric detection usually requires specific filters for the fluorophore of choice.
Each assay has different sensitivity, as it depends on what they are 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.
Samples should be completely homogenized, preferably using a Dounce homogenizer. 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 deproteinating the sample prior analysis using a 10kD spin column (ab93349).
Assay buffer compositions and concentration cannot be publically disclosed as they are propietary information. A MSDS (Material Safety DataSheet) on the components can be found on each product datasheet which contains information on hazardous ingredients and hazard identification.
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 in the range for the kit. Therefore, we encourage users to follow the sample recommendation on the protocol.
At the moment Abcam does not sell separate kit components on a general basis. We understand however that sometimes you might need extract components. If extra components are available for sale, the information will be available on the datasheet.