Ammonia assay kit

Manufactured by Merck Group

Sourced in United States, Germany

The Ammonia Assay Kit is a laboratory tool designed to quantify the concentration of ammonia in various sample types. It provides a colorimetric method for the determination of ammonia levels. The kit includes all the necessary reagents and instructions for performing the assay.

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Close spelling variants of this product

Sigma-Aldrich® Ammonia Assay Kit (4 citations) Ammonia assay kit (AA0100 (2 citations)

68 protocols using ammonia assay kit

Quantifying Urease and Hydrogen Uptake in H. pylori

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Urease activity of whole H. pylori cells was assayed by measuring the ammonia production using the Ammonia-Assay kit (Sigma) as described [13 (link)]. The NH₃ concentration in the supernatant was measured with the Ammonia-Assay kit according to the manufacturer’s (Sigma) instructions. Hydrogen uptake activity was determined spectrophotometrically at 604 nm by following the color change of methyl viologen (MV) from a colorless oxidized form to a dark-violet reduced form as described in [20 (link)]. The data correspond to at least three independent experiments with two technical replicates each time.

Denic M., Turlin E., Michel V., Fischer F., Khorasani-Motlagh M., Zamble D., Vinella D, & de Reuse H. (2021). A novel mode of control of nickel uptake by a multifunctional metallochaperone. PLoS Pathogens, 17(1), e1009193.

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Quantifying Ammonia in Cell Media

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Ammonia levels in cell-conditioned medium was determined by using the ammonia assay kit (Sigma-Aldrich, AA0100) following the manufacturer's protocol. Briefly, cells were grown for several days and medium was collected every other day. 100 μl of cell medium was mixed with 1 ml of ammonia assay reagent and incubated or 5 min at room temperature. Afterwards, 10 μl of glutamate dehydrogenase solution was added. After 5 min absorbance was read at 340 nm in a Glomax multi detection system. In all measurements ammonia production in the absence of cells was subtracted from each experiments.

Polletta L., Vernucci E., Carnevale I., Arcangeli T., Rotili D., Palmerio S., Steegborn C., Nowak T., Schutkowski M., Pellegrini L., Sansone L., Villanova L., Runci A., Pucci B., Morgante E., Fini M., Mai A., Russo M.A, & Tafani M. (2015). SIRT5 regulation of ammonia-induced autophagy and mitophagy. Autophagy, 11(2), 253-270.

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Quantifying Ammonia Production in Bacterial Strains

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Ammonia production in the culture supernatant of wild-type, mutant, and complementary strains was quantified using an Ammonia Assay kit (Sigma-Aldrich Corporation, St. Louis, MO, USA) in accordance with the manufacturer's instructions.

Gao T., Yuan F., Liu Z., Liu W., Zhou D., Yang K., Duan Z., Guo R., Liang W., Hu Q., Tian Y, & Zhou R. (2019). MnmE, a Central tRNA-Modifying GTPase, Is Essential for the Growth, Pathogenicity, and Arginine Metabolism of Streptococcus suis Serotype 2. Frontiers in Cellular and Infection Microbiology, 9, 173.

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Serum Ammonia Measurement Protocol

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Blood samples were placed at room temperature for at least 30 min for coagulation. Serum samples were obtained by centrifugation at 3000 rpm for 15 min at 4°C. The ammonia concentration in the serum was determined using a Sigma-Aldrich Ammonia Assay Kit (AA0100). The procedure was performed according to the manufacturer’s instructions.

Chen Y., Tran H., Saber Y, & Hall F.S. (2020). High ambient temperature increases the toxicity and lethality of 3,4-methylenedioxymethamphetamine and methcathinone. Pharmacology, biochemistry, and behavior, 192, 172912.

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Effluent Nutrient Monitoring in DFR

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Nitrate and ammonia measurements were performed on effluent media collected from the DFR right before biofilm harvest. Effluent media was collected for 5 min before harvest, and the effluent was filtered on 0.2 µm syringe filters (LLG Labware) to remove cells. Subsequently the ammonia concentration in the effluent was measured with the Ammonia Assay Kit (Sigma-Aldrich) according to manufacturers instructions and nitrate was measured with the Nitrite/Nitrate Assay Kit (Sigma-Aldrich).

Herschend J., Damholt Z.B., Marquard A.M., Svensson B., Sørensen S.J., Hägglund P, & Burmølle M. (2017). A meta-proteomics approach to study the interspecies interactions affecting microbial biofilm development in a model community. Scientific Reports, 7, 16483.

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Purification and Detection Reagents

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Blasticidin hydrochloride (10 mg/ml stock solution), hygromycin B (50 mg/ml solution), Geneticin sulfate (75% [wt/vol] purity), SuperSignal West Pico Plus chemiluminescent substrate, 6×His tag monoclonal antibody horseradish peroxidase (His.H8), and mouse antihemagglutinin (anti-HA) monoclonal antibody were from Invitrogen (Thermo Fisher Scientific). l-Histidine, anti-α-tubulin monoclonal antibody, mouse anti-c-Myc, ammonia assay kit, acid-washed glass beads (425 to 600 μm), protease inhibitor cocktail (catalog no. P2815), resazurin, EDAC [1-ethyl-3-(dimethylaminopropyl)carbodiimide] HCl, and rabbit anti-FLAG were from Sigma Chemical Co., and chicken anti-GFP was from Abcam. Guinea pig anti-vacuolar proton pyrophosphatase (VP1) was produced in-house (30 (link)), rabbit anti-pyruvate phosphate dikinase (PPDK) was a gift from Frédéric Bringaud (University of Bordeaux, France), mouse anti-histidine ammonia lyase (HAL) was kindly provided by Ariel Silber (University of São Paulo, Brazil). Aminomethylenediphosphonate (AMDP) was a gift from Eric Oldfield (University of Illinois at Urbana-Champaign), polyP₁₀₀ (i.e., 100 P_i units) was kindly provided by Shiba Toshikazu (RegeneTiss Co., Japan) or was purchased from Kerafast. Polymethacrylate beads (ReliZyme EA113; particle size, S, EC-HA) carrying an ethylenediamino group were purchased from Resindion srl (Milan, Italy).

Mantilla B.S., Azevedo C., Denny P.W., Saiardi A, & Docampo R. (2021). The Histidine Ammonia Lyase of Trypanosoma cruzi Is Involved in Acidocalcisome Alkalinization and Is Essential for Survival under Starvation Conditions. mBio, 12(6), e01981-21.

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mTG-Mediated Gelatin Cross-Linking and Ammonia

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NO gels 2.4% (#1), 4.8% (#2), and 9.6% (#3) were formed by mTG-mediated cross-linking of gelatin. Media supernatants were then collected to determine ammonia concentrations during the cross-linking reaction of test gels (n = 3). Samples were centrifuged for 5 min at 14,000 rpm to remove particles. The ammonia concentration was measured using an ammonia assay kit (Sigma-Aldrich) following the manufacturer’s instructions. Briefly, samples were mixed and reacted with working reagents for 15 min in the dark at room temperature. The fluorescence intensity as an indication of total ammonia concentration was measured at E_x = 360 nm and E_m = 450 nm (BioTek, Republic of Korea).

Kang M.L., Kim H.S., You J., Choi Y.S., Kwon B.J., Park C.H., Baek W., Kim M.S., Lee Y.J., Im G.I., Yoon J.K., Lee J.B, & Sung H.J. (2020). Hydrogel cross-linking–programmed release of nitric oxide regulates source-dependent angiogenic behaviors of human mesenchymal stem cell. Science Advances, 6(9), eaay5413.

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Purification and Activity Assay of PncA

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His-tag Protein Purification Kit (P2226) from Beyotime (Beijing, China) was used to purify PncA. PncA activity was determined by the ammonia production detected by the ammonia assay kit (MAK310) from Sigma-Aldrich. In a typical assay, 100 mM HEPES, pH 7.4, containing 500 μM nicotinamide and 37.5 nM PncA were combined at 27 °C. The addition of enzyme initiated the reaction, and ammonia was detected after 15 min. The amount of conversion of NAM by PncA was calculated by ammonia production. As for the bacterial enzyme assay, lysis of E. coli was used.

Feng S., Guo L., Wang H., Yang S, & Liu H. (2023). Bacterial PncA improves diet-induced NAFLD in mice by enabling the transition from nicotinamide to nicotinic acid. Communications Biology, 6, 235.

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Quantitative Metabolite Monitoring

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Ammonium and glucose in the culture media were quantified at the indicated time points using an ammonia assay kit (Sigma) and a glucose oxidase (GOD) assay (Roche Diagnostics GmbH), respectively and following the manufacturer's instructions.

Santos J., Leitão-Correia F., Sousa M.J, & Leão C. (2014). Ammonium is a key determinant on the dietary restriction of yeast chronological aging in culture medium. Oncotarget, 6(9), 6511-6523.

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Leaky Ammonia Activity Measurement

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To measure the leaky (unproductive) ammonia activity, previously reported method was used (27 (link)). The reaction mixture mentioned above for glutamate production, containing 1 μM of the test enzyme, was supplemented with ammonia assay reagent [containing a mixture of α-ketoglutarate and NADPH [reduced form of nicotinamide adenine dinucleotide phosphate (NADP⁺)] and with l-glutamate dehydrogenase enzyme provided in the ammonia assay kit from Sigma-Aldrich. NADPH oxidation to NADP⁺ was monitored by recording the absorbance at 340 nm.

Sharma N., Ahalawat N., Sandhu P., Strauss E., Mondal J, & Anand R. (2020). Role of allosteric switches and adaptor domains in long-distance cross-talk and transient tunnel formation. Science Advances, 6(14), eaay7919.

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