Product Overview :
The Glucose Uptake Assay Kit is a non-radioactive, plate-based, homogeneous bioluminescent method for measuring glucose uptake in mammalian cells based on the detection of 2-deoxyglucose-6-phosphate (2DG6P). When 2-deoxyglucose (2DG) is added to cells, it is transported across the membrane and rapidly phosphorylated in the same manner as glucose. However, enzymes that further modify glucose-6-phosphate (G6P) cannot modify 2DG6P, and thus this membrane-impermeable analyte accumulates in the cell. After a brief period of incubation, an acid detergent solution (Stop Buffer) is added to lyse cells, terminate uptake and destroy any NADPH within the cells. A high-pH buffer solution (Neutralization Buffer) is then added to neutralize the acid. A Detection Reagent containing glucose-6-phosphate dehydrogenase (G6PDH), NADP+, Reductase, Ultra-Glo™ Recombinant Luciferase and proluciferin substrate is added to the sample wells. G6PDH oxidizes 2DG6P to 6-phosphodeoxygluconate and simultaneously reduces NADP+ to NADPH. The Reductase uses NADPH to convert the proluciferin to luciferin, which is then used by Recombinant Luciferase to produce a luminescent signal that is proportional to the concentration of 2DG6P.
Kit Components :
The system contains sufficient reagents to perform 50 reactions in 96-well plates (50μl of sample + 25μl of Stop Buffer + 25μl of Neutralization Buffer + 100μl of 2DG6P Detection Reagent). Includes:
• 5ml Luciferase Reagent
• 15ml Stop Buffer
• 15ml Neutralization Buffer
• 50μl NADP+ (20mM)
• 125μl Glucose-6-Phosphate Dehydrogenase (G6PDH)
• 25μl Reductase
• 55μl Reductase Substrate
• 250μl 2-deoxyglucose (2DG, 100mM)
• 50μl 2DG6P Standard (1mM)
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