Promega

 

Glucose Uptake-Glo™ Assay

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Glucose Uptake-Glo™ Assay 

 

 

The Glucose Uptake-Glo™ Assay 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 Ultra-Glo™ Recombinant Luciferase to produce a luminescent signal that is proportional to the concentration of 2DG6P.

 

Features - Benefits

 

 - Use a Non-Radioactive Assay: The assay is based on the same principal as the radioactive approach, but no radioactivity is required.

 - Follow a Simple and Homogeneous Protocol: After addition of 2DG, there are no wash steps—all steps are additions.

 - Achieve Sensitivity with Broad Linearity: The Glucose Uptake-Glo™ Assay can detect 0.5 to 30µM 2DG6P and generates a signal-to-background ratio >3 with as few as 5,000 cells.

 - Automate your Workflow: The add-and-read format is compatible with automated and high-throughput workflow; reactions are scalable for use in 96- and 384-well plates.

 - Get Reliable and Reproducible Results: The Glucose Uptake-Glo™ Assay yields Z´ factors >0.5.

 

Applications

 

Assay glucose uptake in mammalian cells, including insulin-sensitive cell types and cancer cells. 

 

Notes 


Using 50µl of sample + 25µl of Stop Buffer + 25µl of Neutralization Buffer + 100µl of 2DG6P Detection Reagent in a 96-well plate: Cat.# J1341 provides sufficient reagents to perform 50 assays; Cat.# J1342, 100 assays; Cat.# J1343, 500 assays. 



  제품 background
  • Glucose metabolism은 많은 생명체에서 이루어지는 주요 대사 반응으로 insulin-stimulated glucose uptake이 부족하게 되면 type 2 diabetes가 발생하는 것과 관계되며, 많은 양의 glucose uptake가 발생하는 것은 high glycolytic rate를 의미하기 때문에 cancer와 연관되게 됩니다. 따라서 glucose uptake를 연구함으로써 당뇨병과 암의 치료제의 효능을 평가할 수 있게 됩니다.


  제품 원리
  • 2-deoxyglucose (2DG)를 세포에 처리하면 세포로 흡수되며 흡수된 2DG는 2DG6P (2-dexoyglucose-6-phosphate)로 대사가 일어나게 됩니다. 이는 다음 단계로의 대사가 일어나지 못하고 세포 내에 축적되게 됩니다. 이를 측정하므로 세포 내로 uptake된 glucose 양을 측정하는 원리입니다.




  제품 특장점
  • Non-radioactive 방법: 이 분석 방법은 방사성 방법과 동일한 원리를 기반으로한 발광 기반의 분석 방법입니다.
  • 간단하고 동질적인 (homogeneous) 실험 방법: 2DG를 첨가한 후에 washing 단계가 없는 시약 첨가의 단계만 있습니다.
  • 광범위한 linearity와 민감도: 0.5 ~ 30μM 2DG6P 검출 가능하며 5000개 세포에서 signal-to-background > 3 입니다.
  • 자동화와 호환 가능: “Add-and-read” 형태로 자동화와 HTS (96- & 384-well plate)에 적용 가능합니다.


  경쟁사 및 경쟁 제품
  • homebrew 방식이나 방사선 동위원소 기법이 gold standard 방법으로 가장 많이 사용됨, 형광 또는 흡광 기법을 이용한 commercial product가 여러 회사 (Abcam, Sigma 등)에서 판매 중이나 감도가 떨어져 신뢰성 높은 세포 내에서의 glucose uptake결과를 측정하기 어렵습니다. 잘 알고 계신 것처럼 RI 기법은 민감도가 높아 선호되는 기법입니다. 발광 기법도 유사한 민감도를 제공합니다.



Keyword: Glucose update, non-radioactive glucose uptake, glucose metabolism, energy metabolism, oncology, 대사 질환, 당뇨병, 비만



Target customer


 - Customer type - Academia, Pharma/Biotech

 - Application - Metabolism, diabetes, oncology, cell differentiation (iPS)

 

 

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