Glutamine/Glutamate-Glo™ Assay

How the Assay Works

The assay is based on the conversion of glutamine to glutamate by glutaminase enzyme. Next, glutamate oxidation and NADH production are coupled with a bioluminescent NADH detection system. Glutamate dehydrogenase uses glutamate and NAD+ to produce α-ketoglutarate and NADH. In the presence of NADH, a pro-luciferin Reductase Substrate is converted by Reductase to luciferin that is then used by Ultra-Glo™ Recombinant Luciferase to produce light. 

This assay requires two steps: i) glutamine conversion to glutamate by Glutaminase; and ii) glutamate detection with the Glutamate Detection Reagent. When Glutamate Detection Reagent, which contains glutamate dehydrogenase (GlutDH), NAD+, Reductase, Reductase Substrate and Luciferase, is added to a sample containing glutamate at a 1:1 ratio, the enzyme-coupled reactions start and run simultaneously. The luminescent signal is proportional to the amount of glutamate and increases until all glutamate is consumed, at which time a stable luminescent signal is achieved.

When using this assay, both glutamine and glutamate will be measured. There is no need to use a separate assay for glutamate. For samples that contain both glutamate and glutamine, the light signal will be proportional to the starting concentration of total glutamine plus glutamate. Therefore a second reaction without the Glutaminase enzyme is needed to measure the glutamate-only concentration. Glutamine levels can be calculated by subtracting the glutamate-only signal from the total glutamine plus glutamate signal.

Glutamine and Glutamate Titration Curves

Advantages

Quickly Measure Metabolites in a Variety of Sample Types: Measure in media, cells, tissue and plasma. The assays require minimal preparation steps with no need for centrifugation and spin columns.

Conduct easy in-well processing for intracellular detection.

Measure Across a Wide Range of Concentrations: Broad linearity up to 3 logs enables simple sample measurement.

Detect Small Changes: Wide assay window with S/Bmax > 300 allows better discrimination of small changes in metabolite levels compared to colorimetric and fluorometric assays.

Gain More Information Per Sample: The assays are amenable to measuring multiple metabolites from the same sample. Multiplex with cell viability assays for normalization.

Glutamine Consumption and Glutamate Secretion by SKOV-3 Cells