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Product Details
| Cat # +Size | K666-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em = 535/587 nm) |
| Species Reactivity | Mammalian |
| Applications | This easy to use non-radioactive kit can detect glucose uptake as low as 50 pmol/well and can be used for a variety of cell types. |
| Features & Benefits | • Simple procedure • Fast and convenient • The assay is easy, non-radioactive, and highly sensitive |
| Kit Components | • Extraction Buffer • Neutralization Buffer • 2-Deoxyglucose (2-DG, 10 mM) • 2-DG Uptake Assay Buffer • Enzyme Mix (Lyophilized) • PicoProbe™ (in DMSO) • 2-DG6P Standard (Lyophilized) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Glucose uptake is an important biological process for studying cell signaling and glucose metabolism. Among many different methods available for measuring glucose uptake, 2-deoxyglucose (2-DG) has been widely used because of its structural similarity to glucose. As with glucose, 2-DG can be taken up by glucose transporters, and metabolized to 2-DG-6-phosphate (2-DG6P). 2-DG6P, however, cannot be further metabolized, and thus accumulates in the cells. The accumulated 2-DG6P is, therefore, directly proportional to 2-DG (or glucose) uptake by cells. In BioVision’s glucose uptake fluorometric assay kit, the accumulated 2-DG6P is enzymatically oxidized and coupled to BioVision’s PicoProbe, which generates fluorescence in the presence of NADPH. This easy to use non-radioactive kit can detect glucose uptake as low as 50 pmol/well and can be used for a variety of cell types.
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I have a question in regards to your kits to measure Glucose uptake by Colorimetry and Fluorometry (K676-100 and K666-100). Fluorometric methods are usually more sensitive than colorimetric ones; however, according to the datasheets, the colorimetric kit can measure glucose as low as 10 pmol/well, whereas the fluorometric one can detect glucose uptake as low as 50 pmol/well. Could you please explain this?
Yes, in this case the colorimetric assay is more sensitive than the fluorimetric assay, since the colorimetric assay includes a Recycling Amplification step, which amplifies the signal many folds. This step is not included in the fluorometric assay and therefore it is like an end point one cycle assay without any amplification of the absolute signal.
I want to use this kit to detect the ability of glucose uptake in cells containing the GFP protein. Is this kit suitable?
Yes, you can use this kit with your cells with some optimization. GFP detection is ideally done at Ex/Em 488/507 nm and this kit utilizes Ex/Em = 535/587 nm, therefore there should not be a significant overlap.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
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 expt with multiple sample volumes to determine the optimal volume 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.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which 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.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your 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.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Na, Yi Rang et al. (2016) GM-CSF Induces Inflammatory Macrophages by Regulating Glycolysis and Lipid Metabolism, J Immunol. 2016 Nov 15;197(10):4101-4109.
Wang et al., JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α–mediated glucose metabolism. PNAS, Dec 2013; 10.1073/pnas.1311249111.
For more citations of this product click here
