Glucose Uptake Assay Kit – Information
Assay Genie’s 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 Ex/Em = 530/585 nm proportional to the concentration of 2-DG6P.
Determination of glucose uptake in whole cells and evaluation of effects of ligands or drugs on glucose transport.
Glucose Uptake Assay Kit – 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.
Glucose Uptake Assay Kit – Data Sheet
|Kit Includes||Assay Buffer: 10 mL G6P Reagent: 1.5 mL Enzyme A: 120 µL Enzyme B: 120 µL G6PDH Enzyme: 120 µL NADP: 120 µL 2-DG Substrate: 1.2 mL Probe: 750 µL 2-DG6P Standard: 120 µL NADP/NADPH Extraction Buffers: each 12 mL|
|Kit Requires||Triton X-100 ; Phosphate Buffered Saline; black cell culture 96-well plate; deionized or distilled water; pipetting devices; cell culture incubators; centrifuge tubes; fluorescence plate reader capable of reading at lex/em = 530/585 nm|
|Method of Detection||FL530/585nm|
|Detection Limit||0.1 μM|
|Protocol Length||Approximately 2 hrs|
|Storage||Store all reagents at -20°C.|
|Shelf Life||6 months|
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.