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Product Details
| Cat # +Size | K676-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (412 nm) |
| Species Reactivity | Mammalian |
| Applications | This easy to use non-radioactive kit is highly sensitive and can detect glucose uptake as low as 10 pmol/well. |
| 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) • Assay Buffer • Enzyme Mix (lyophilized) • Recycling Mix (lyophilized) • 2-DG6P Standard (lyophilized) • Glutathione Reductase (lyophilized) • Substrate-DTNB (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 directly proportional to 2-DG (or glucose) uptake by cells. In BioVision’s glucose uptake colorimetric assay kit, the 2-DG6P is oxidized to generate NADPH, which can be determined by an enzymatic recycling amplification reaction. This easy to use non-radioactive kit is highly sensitive and can detect glucose uptake as low as 10 pmol/well.
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In the step VIII - 3b, some sample is lost in the heating step (at 90°C for 40 min). Do you have any solution for this problem? What equipment do you recommend, for example, water bath or heating block? When I used water bath, the plate was bent from the heat. If the plate is bent, Should I transfer the sample to a new plate?
We would recommend using a heating block or an incubator and not a water bath for heating the samples. You can also use a heat resistant 96 well plate which can withstand heat up to 120°C. If the plate is bent, please transfer samples to a new plate. Please use a lid to cover the plate to avoid loss of sample through evaporation.
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 have some questions about your glucose uptake colorimetric assay kit (ref K676-100). I'm working on human myotubes and I would to know if it's possible to start from 3.5cm petri dish and then continue into 96-well plate after the lysis step? If yes, do you have an idea on volumes of buffers/reagents that I need to use ?
Yes, you can certainly do that. You have to increase the volumes of all reagents used such that the cells are covered and are not exposed to the air to dry out. For eg., for the serum starvation, instead of using the 100 µl media for covering cells in the 96 well plate, you could use about 2 ml media for the 3.5 cm plate.
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.
Instead of KRPH, can we use media without glucose or serum for starvation?
KRPH is suggested in the protocol because the composition of the buffer is closest to the physiological levels without any nutrients in it. Most media will have pyruvate or other substrate that can lead to glucose generation. Hence, KRPH is recommended.
Have you used this kit on organoids cultured in matrigel?
No, we have not used the kit on organoids in matrigel. We would assume that you would need to release the cells from the matrigel through a digestion step before using it with this kit.
What are the specific conditions for freezing and thawing in sample processing? What is the processing temperature and time?
You can do overnight freezing at -80°C and thawing the next day. This only needs to be done once.
The examples in the instructions are fat cells. Is this KRPH buffer treatment for fat cells or is it necessary for all cells to be processed?
You can use buffers that are recommended for your cell type. You do not have to use KRPH buffer. However, KRPH contains components that are physiologically relevant for the cells.
Why do I need insulin to stimulate the cells? Can I do the assay without insulin stimulation?
Insulin will stimulate glucose transporter (such as GLUT 2) and translocate the glucose transporter to the surface of the membrane making the cells more responsive to glucose. You can try both, with and without insulin to see the response from your sample type.
Could you please share the protocol for cell lysate preparation?
Homogenize 1X10^6 cells in 100 µL of Extraction Buffer using a dounce homogenizer or sonicator on ice. Once lysis is completed, centrifuge the lysate at 10,000 g for 5 min at 4°C and use the supernatant for the assay.
Isabelle K. Vila, A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity. JCI Insight, July 2019; 31292296.
Wei Jiang, The PIK3CA E542K and E545K mutations promote glycolysis and proliferation via induction of the β-catenin/SIRT3 signaling pathway in cervical cancer. J Hematol Oncol, Dec 2018; 30547809.
Yunjie Xu, et al., (2018) ABT737 reverses cisplatin resistance by targeting glucose metabolism of human ovarian cancer cells, International Journal of Oncology, Jul. 2018, 30015875
Gu Z et al., (2017) NEK2 Promotes Aerobic Glycolysis in Multiple Myeloma Through Regulating Splicing of Pyruvate Kinase, J Hematol Oncol, 2017, 13;10(1):17
Sun, Xianghui et al. (2017) MicroRNA-143 suppresses oral squamous cell carcinoma (OSCC) cell growth, invasion and glucose metabolism through targeting Hexokinase2, Biosci Rep. 2017 Jun 8;37(3).
For more citations of this product click here
