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BioAssay Systems/EnzyFluo™ Glucose Uptake Assay Kit/100 tests/EFGU-100

EnzyFluo™ Glucose Uptake Assay Kit

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ProtocolSDS

Application

Determination of glucose uptake in whole cells and evaluation of effects of ligands or drugs on glucose transport.

Key Features

Safe. No radioactive material is used.
Sensitive and Accurate. Detection limit of 0.1 µM and linearity up to 5 µM 2-DG6P.
Simple and Convenient. Can be automated as a medium throughput assay for glucose transport in cells.

Method

FL530/585nm

Samples

Cell culture

Species

Mammalian

Size

100 tests

Detection Limit

0.1 μM

Shelf Life

6 months

More Details

Glucose Uptake has a variety of methods and transporters, and depends upon the metabolic demand of the cell type and availability of glucose. There are over ten different facilitated diffusion glucose transporters which transports glucose down its concentration gradient without ATP hydrolysis. In the kidneys, secondary active transport is used to uptake Glucose against its concentration gradient to ensure that very little glucose is excreted in urine. BioAssay Systems fluorescent cell-based glucose uptake assay uses 2-deoxyglucose (2-DG), a widely used glucose analog because it can be taken up by glucose transporters and metabolized by endogenous hexokinase into 2-deoxyglucose 6-phosphate (2-DG6P). 2-DG6P accumulates intracellularly because it is not a suitable substrate for phosphoglucose isomerase, the next step in glycolysis. The cells are lysed, and excess NADP and glucose 6-phosphate dehydrogenase (G6PDH) is added to metabolize 2-DG6P and generate a molar equivalent amount of NADPH. The NADPH is then measured using a G6PDH recycling reaction to amplify the signal and generate a fluorescent signal measureable at λex/em = 530/585 nm proportional to the concentration of 2-DG6P.

I don"t have the correct wavelength filter, will the assay still work?

Typically if you are within ± 20 nanometers the assay will still work, but have slightly reduced sensitivity. Some assays have a narrower peak than others, call our tech support for a spectrum to verify.

What is the shelf life of this kit?

The shelf life is 6 months from the date you received the kit. The reagents are ready to use. No reagent reconstitution is needed.

Does the assay kit work in particular cells?

Yes, this assay works in all mammalian culture cells. We have not tested this kit in other cells, such as bacteria, yeast cells etc.

How do I normalize the results?

One method to normalize the results is to divide the glucose uptake activity against total number of cells or total cellular protein in the assay well. This would require a protein determination kit (e.g. BioAssay Systems" QCPR-500 or QFPR-200).

Can I store unused reagents for future use?

Yes, unused reagents can be stored according to the assay protocol. Repeated freeze/thaw cycles of reagents should be avoided.

Do I need to use a standard curve with each assay run?

Yes, it is highly recommended.For more detailed product information and questions, please feel free to Contact Us. Or for more general information regarding our assays, please refer to our General Questions.

Liu, Meihan, et al (2019). Activation of AMPK by metformin promotes renal cancer cell proliferation under glucose deprivation through its interaction with PKM2. International Journal of Biological Sciences 15.3: 617-627. Assay: Glucose uptake in human cells. Park, Seong-Hoon, et al(2016). SIRT2-mediated deacetylation and tetramerization of pyruvate kinase directs glycolysis and tumor growth. Cancer research 76.13: 3802-3812. Assay: Glucose uptake in human cells. Tunduguru, Ragadeepthi (2016). Regulation of skeletal muscle insulin sensitivity by PAK1. Diss. Assay: Glucose uptake in rat cells. To find more recent publications, pleaseclick here.

If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.Simply send us your samples:- Fast turnaround - Quality data - Low costPlease email or call 1-510-782-9988 x 2 to request assay service.

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