The available kits are too expensive for the number of samples we need to do.
See Van Kuilenburg et al (1994) describing a spectrophotometric CTP synthetase assay. In your case, you only need the final step of their assay to measure your analyte (glutamate) by adding glutamate dehydrogenase and oxidized acetyl pyridine adenine dinucleotide (APAD+, 1 mM) and measuring the absorbance rate at 365 nm.
Normally the glutamate dehydrogenase reaction is strongly favored in the forward direction (alpha-ketoglutarate + ammonium + NADH --> glutamate + NAD+). The interesting thing about this assay is that substituting APAD+ for the nicotinamide cofactor allows the reaction to proceed in the opposite direction.
The bovine liver glutamate dehydrogenase from Sigma (cat. no. G-7882) is nearly ammonia-free (
An easy way to reduce the cost of using kits is to reduce the size of the assay. If the right equipment is available, absorbance, fluorescence, and luminescence assays can be performed in 384-well plates in much smaller volumes that most kits call for, allowing the kit reagents to be used for far more measurements. This requires a plate reader with the necessary capabilities, and is greatly aided by multichannel pipettors.
See Van Kuilenburg et al (1994) describing a spectrophotometric CTP synthetase assay. In your case, you only need the final step of their assay to measure your analyte (glutamate) by adding glutamate dehydrogenase and oxidized acetyl pyridine adenine dinucleotide (APAD+, 1 mM) and measuring the absorbance rate at 365 nm.
Normally the glutamate dehydrogenase reaction is strongly favored in the forward direction (alpha-ketoglutarate + ammonium + NADH --> glutamate + NAD+). The interesting thing about this assay is that substituting APAD+ for the nicotinamide cofactor allows the reaction to proceed in the opposite direction.
The bovine liver glutamate dehydrogenase from Sigma (cat. no. G-7882) is nearly ammonia-free (
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