2-Deoxyglucose (2DG) Uptake Measurement Kit

Measurement of 2-deoxyglucose (2DG) uptake in tissues and cells is a reliable approach for estimating glucose uptake and thereby to explore the regulation of glucose metabolism and mechanisms of insulin resistance. While assays employing radioisotope-labeled 2DG are often used to measure 2DG uptake in vivo and in vitro, the use of radioisotopes is not possible without problems including permitting, handling, and disposal. Furthermore, in actual practice, radioactive assay protocols for 2DG uptake require a corrective separation step to account for labeled 2DG that remains in extracellular fluids and can lead to substantial variation. Such problems are obviated by the enzymatic method of 2DG detection employed by this kit, based on published methods (Saito K and Minokoshi Y, et al. Analytical Biochem 412: 9-17, 2011).

Features

• No need for radioisotopes (RIs) or radiation counting instrumentation
  
• Photometric readout on standard microplate readers
  
• Direct measurement of 2DG6P accumulation in cells
  
• High sensitivity achieved by a recycling enzymatic amplification reaction
  

Assay principle in five key steps

1. Oxidation of glucose-6-phosphate (G6P) with a low concentration of G6P dehydrogenase (G6PDH) with NAD+ to eliminate endogenous G6P in target cells
   
2. Elimination of NAD(P)H with HCl, which removes endogenous NAD(P)H as well as NADH produced in step 1
   
3. Generation of NADPH through oxidation of 2DG6P in cells with a high concentration of G6PDH, the generated NADPH being used for quantification of 2DG6P
   
4. Elimination of NAD(P)+ and G6PDH that remains after Step 2 with NaOH
   
5. Recycling amplification reaction for the small amount of NADPH generated, and quantification of 2DG6P using a photometric microplate reader at 420 nanometers wavelength