A two-step protocol was used to assay glutaminase activity, as previously described28 (link). In the first reaction, glutaminase catalyses the hydrolysis of glutamine to glutamate, and in the second reaction glutamate dehydrogenase catalyses the oxidative deamination of glutamate to form α-ketoglutarate and NADH, which can be measured through its absorbance at 340 nm. Briefly, mitochondria (10 μg total protein) were added to 105 μl of reaction mix 1 (20 mM glutamine, 0.2 mM EDTA, 50 mM Tris-acetate, pH 8.6). Samples were rotated at 37 °C for 45 min. The reaction was then quenched by adding 10 μl of 3 M HCl, and samples were placed on ice. Next, 20 μl of reaction mix 1 was added to 200 μl of reaction mix 2 (1 unit bovine liver glutamate dehydrogenase (Sigma-Aldrich), 80 mM Tris-HCl pH 9.4, 200 mM hydrazine, 0.25 mM ADP, 2 mM NAD). The samples were mixed and incubated for 1 h at room temperature. The A340 was then determined against a blank in which a heat-inactivated mitochondrial sample was added to reaction mix 1. A standard curve was also prepared by adding known amounts of glutamate to reaction 2. This allowed the amount of glutamate generated during reaction 1 to be determined. All assays were carried out in triplicate, and the mean and s.d. calculated.
Bovine liver glutamate dehydrogenase
Manufactured by Merck Group
Sourced in United States
Bovine liver glutamate dehydrogenase is an enzyme isolated from the liver of cattle. It catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia, playing a role in amino acid metabolism.
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Lab products found in correlation
Asparagine, by Merck Group (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
Cesium chloride, by Merck Group (1 mentions)
Dialyzed fetal bovine serum, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
Aspartate, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Cb 839, by Selleck Chemicals (1 mentions)
Cyclin a, by Santa Cruz Biotechnology (1 mentions)
Dimethyl α ketoglutarate, by Merck Group (1 mentions)
K2hpo4, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
6 diazo 5 oxo l norleucine, by Merck Group (1 mentions)
Endothelial cell growth factor, by BD (1 mentions)
5 and 6 chloromethyl 2 7 dichlorodihydrofluorescein diacetate acetyl ester cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Ng nitro l arginine methyl ester l name, by Selleck Chemicals (1 mentions)
20 ci mmol, by PerkinElmer (1 mentions)
Rainbow molecular weight markers, by GE Healthcare (1 mentions)
6 protocols using bovine liver glutamate dehydrogenase
1
Glutaminase Activity Assay Protocol
2
Metabolic Regulation of Cell Cycle and Oxidative Stress
3
Mitochondrial Glutaminase Activity Assay
4
Glutaminase Enzyme Activity Assay
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Glutaminase enzyme activity of recombinant human glutaminase was assessed by glutaminase activity assay using a two-step assay [36 (link), 37 (link)]. Briefly, protein concentrations in the lysates were tested using BCA Protein Assay Kit (Pierce). In the first step, fifty milligrams of diluted glutaminase was added to 100 μL of initial assay mix, the mix contained 50 mmol/L glutamine, 0.15 mol/L phosphate, 0.2 mmol/L EDTA, and 50 mmol/L Tris-acetate, pH 8.6, incubated at 37 °C for 30 min. 10 μL of 3 N hydrochloric acid (HCl) was added to inactivate the glutaminase activity and stop the reaction. In the second step, 1 mL of the second reaction mixture was added, which contained 0.4 mg of purified bovine liver glutamate dehydrogenase (Sigma-Aldrich, St. Louis, MO, USA), 0.08 mol/L Tris-acetate, pH 9.4, 0.2 mol/L hydrazine, 0.25 mmol/L ADP (adenosine 5'-diphosphate sodium salt), and 2 mmol/L NAD (β-nicotinamide adenine dinucleotide hydrate). The samples were mixed and incubated for 30 min at room temperature. 100 μL of reaction was used for measurement, the absorbance at 340 nm, and glutamate concentration was determined using a standard curve of 20, 10, 5, 2.5, 1.25, 0.625, and 0.0 mmol/L glutamate, along with negative controls.
5
Mitochondrial Glutaminase Activity Assay
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A two-reaction protocol was used to measure mitochondrial glutaminase activity. Mitochondria (5 μg total protein) were added to 105 μL of Reaction Mix 1 (20 mM glutamine, 0.2 mM EDTA, 50 mM Tris-acetate pH 8.6), supplemented with 10 μM BPTES when appropriate, and samples were incubated at 37°C for 40 min. The reaction was then quenched by addition of 10 μL of 2.4 M HCl, and samples placed on ice. Next, 20 μL of quenched Reaction Mix 1 was added to 200 μL of Reaction Mix 2 (1 unit bovine liver glutamate dehydrogenase (Sigma-Aldrich), 80 mM Tri-HCl pH 9.4, 200 mM hydrazine, 0.25 mM ADP, 2 mM NAD) and samples were incubated for 1 h at room temperature. The absorbance at 340 nm was then measured against a matched sample in which heat-inactivated mitochondria (immersed in boiling water for 5 min) were used. A standard curve was prepared using given concentrations of glutamate in Reaction Mix 2, allowing the amount of glutamate produced in Reaction 1 to be determined.
6
Glutaminase Activity Assay Protocol
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Highly concentrated whole cell lysates were collected from flasks and subjected to GLS activity assay using a two-step assay [27 (link), 47 (link)]. Briefly, protein concentrations in the lysates were tested by using BCA Protein Assay Kit (Pierce). All samples were normalized to same concentration. In the first step, 50 mg of protein were added to 100 μl of initial assay mix. The mix contains 50 mM glutamine, 0.15 M phosphate, 0.2 mM EDTA, and 50 mM Tris-acetate. The PH value of the mix was adjusted to 8.6 and incubated at 37 °C for 30 min. Ten microliters of 3 N hydrochloric acid was added to inactivate the glutaminase activity and stop the reaction. In the second step, 1 ml of the second reaction mix was added, which contained 0.4 mg of purified bovine liver glutamate dehydrogenase (Sigma-Aldrich, St. Louis, MO, USA), 0.08 M Tris-acetate at pH 9.4, 0.2 M hydrazine, 0.25 mM adenosine 5′-diphosphate sodium salt, and 2 mM β-nicotinamide adenine dinucleotide hydrate. The samples were mixed and incubated for 30 min at room temperature. One hundred microliters of the reaction was used for measurement, absorbance was determined at a wavelength of 340 nm, and glutamate concentration was determined using a standard curve of 10, 5, 2.5, 1.25, 0.625, and 0.0 mM glutamate, along with negative controls.
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