Ammonia Assay Kit

Applikation: Biochemisches Assay (BCA)

Produktnummer: ABIN411726

Produktbeschreibung

Beschreibung: Ammonia is an important source of nitrogen for living systems. Nitrogen is required for the synthesis of amino acids, which are the building blocks of protein. Ammonia is a metabolic product which is created through amino acid deamination. It plays an important role in both normal and abnormal animal physiology such as acid/base balance. BioVision provides a rapid, simple, sensitive, and reliable assay suitable for research and high throughput assay of Ammonia. In the assay, Ammonia is converted to a product that reacts with the OxiRed probe to generate color (λ max = 570 nm) which can be easily quantified by plate reader. The kit can detect 1 nmol (~20 μM) of ammonia, much more sensitive than measuring NADPH based ammonia assay.

Anwendungen

Protokoll: 1. Standard Curve Preparation: Dilute the Ammonium Chloride standard solution to 1 mM by adding 10 μl of the 100 mM Ammonium Chloride Standard to 990 μl of ddH 2 O, mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the Ammonium Chloride Standard. 2. Sample Preparations: Tissues (20 mg) or cells (2x10 6 ) can be homogenized in 100 μl Assay Buffer, centrifuge at 13,000 x g for 10 minutes to remove insoluble material. Liquid sample can be tested directly. Add 2-50 μl sample to 96 well plate, bring the volume to 50 μl/well with Assay Buffer. For unknown samples, we suggest testing several different doses of samples to make sure the readings are within the standard curve range .

Applikationshinweise: Note: Pyruvate in samples will interfere with the assay. If significant amount of pyruvate is suspected in your sample, set a Sample Control as in step 3. The pyruvate reading can be subtracted from sample readings. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix. Sample Sample Control Ammonia Assay Buffer 42 μl 44 μl OxiRed Probe 2 μl 2 μl Enzyme Mix 2 μl 2 μl Developer 2 μl 2 μl Converting Enzyme 2 μl 0 μl Add 50 μl of the Reaction Mix to each well containing the NH 4 Cl Standard and test samples. Add 50 μl Sample Control Mix to Sample Control. Mix well. Incubate the reaction for 60 min at 37°C, protect from light. 4. Measurement: Measure O.D. 570 nm in a micro plate reader. 5. Calculation: Correct background by subtracting the value derived from the 0 NH 4 Cl from all readings (The background reading can be significant and must be subtracted from readings). Subtract the Sample Control readings from sample readings. Plot NH 4 Cl standard Curve, NH 4 Cl concentrations of the samples can then be calculated: C = S a /S v nmol/μl or mM, where S a is the sample amount (in nmol) from standard curve. S v is the sample volume (μl) added into the wells. NH 4 + Molecular Weight is 18.04 g/mol.

Beschränkungen: Nur für Forschungszwecke einsetzbar