Ammonia Assay Kit

Produktnummer ABIN1000323

Produktdetails

Target: Ammonia

Detektionsbereich: 24-1000 μM

Untere Nachweisgrenze: 24 μM

Applikation: Biochemical Assay (BCA)

Proben: Serum, Plasma, Urine, Saliva, Cell Culture Supernatant

Produktmerkmale: High sensitivity and wide linear range. Use 20 µL sample. Linear detection range 24 to 1000 µM ammonia.
Homogeneous and simple procedure.
Simple mix-and-measure procedure allows reliable quantitation of NH3 within 30 minutes.

Bestandteile: Assay Buffer: 20 mL. Enzyme: 120 µL. Ketogutarate: 120 µL. Standard: 400 µL 20 mM NH4Cl. NADH Reagent: 1 mL.

Benötigtes Material: Pipeting devices, and clear flat-bottom 96-well plates and optical density plate reader for colorimetric assays, black flat-bottom 96-well plate and fluorescence intensity plate reader for fluorimetric assays.

Anwendungsinformationen

Applikationshinweise: Direct Assays: NH3 in biological samples (e.g. serum, plasma, urine, saliva, cell culture etc).

Protokoll: Colorimetric Procedure
1. Standards and Samples. Equilibrate all components to room temperature. Briefly centrifuge tubes before opening. Prepare a 1000 µMNH3 Standard Premix by mixing 15 µL of the 20 mM Standard and 285 µL dH2O. Transfer 20 µL standards into separate wells of a clear, flat-bottom 96- well plate. Transfer 20 µL of each sample into two separate wells, one serving as a sample blank well (RBLANK) and one as a sample well (RSAMPLE).
2. Enzyme Reaction. For each standard and sample well, prepare Working Reagent by mixing 180 µL Assay Buffer, 1 µL Enzyme, 8 µL NADH Reagent and 1 µL Ketoglutarate. Add 180 µL Working Reagent to the four Standards and the Sample Wells. Prepare blank control reagent by mixing 180 µL Assay Buffer, 8 µL NADH Reagent and 1 µL Ketoglutarate (No Enzyme). Add 180 µL Blank control reagent only to the Sample Blank Wells. Tap plate to mix. Incubate 30 min at room temperature.
3. Read OD340nm. Fluorimetric Procedure The fluorimetric procedure is the same as for the colorimetric assay, except that a black, flat-bottom 96-well plate is used. After incubation for 30 min at room temperature, read fluorescence intensity at ex = 350-360 nm and em = 450 nm.

Aufbereitung der Proben:

Solid samples can be extracted by homogenization in distilled water (dH2O) and filtered, centrifuged or, if necessary, deproteinized to remove any undissolved material. Samples should be clear and colorless with pH adjusted to 7 -
8. Serum and plasma samples can be assayed directly. Cell culture media should be diluted 5-10 fold in dH2O prior to assay.

Ergebnisberechnung:

Subtract the standard values from the blank value (#4) and plot the OD or F against standard concentrations.
Conversions: 1000 µM NH3 equals 1.7 mg/dL or 17 ppm.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Referenzen

Rauner, Leviav, Mavor, Barash: "Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice." in: PLoS ONE, Vol. 8, Issue 6, pp. e68637, (2013) (PubMed).

Núñez, Leme, de Almeida Parizotto, Chagas, de Rezende, da Costa, Monteiro, Boldorini, Jorge, Astray, Pereira, Caricati, Tonso: "Influence of aeration-homogenization system in stirred tank bioreactors, dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism." in: Cytotechnology, (2013) (PubMed).

Antigendetails

Target: Ammonia

Substanzklasse: Anorganic

Hintergrund: Quantitative determination of ammonia/ammonium concentration by colorimetric (340nm) or fluorimetric (360nm/450nm) methods.

Ammonia (NH3) or its ion form ammonium (NH4 + ) is an important source of nitrogen for living systems. It is synthesized through amino acid metabolism and is toxic when present at high concentrations. In the liver, ammonia is converted to urea through the urea cycle. Elevated levels of ammonia in the blood (hyperammonemia) have been found in liver dysfunction (cirrhosis), while hypoammonemia has been associated with defects in the urea cycle enzymes (e.g. ornithine transcarbamylase). Simple, direct and automation-ready procedures for measuring NH3 are popular in research and drug discovery. This ammonia assay is designed to directly measure NH3 and NH4 + . In this assay, NADH is converted to NAD + in the presence of NH3, ketoglutarate and glutamate dehydrogenase. The decrease in optical density at 340 nm or fluorescence intensity at gamma em/ex = 450/360 nm is directly proportionate to the NH3 concentration in the sample.