Ammonia Assay Kit

(Catalog #K370-100; 100 assays; Store kit at -20^oC)

1. 
   

   Introduction:

   
   

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 like normal animal acid/base balance. BioVision developed a rapid, simple, sensitive, and reliable assay suitable for research and high throughput assay of Ammonia. In the assay, Ammonia reacts as substrate with compounds in the presence of enzymes to form 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 M) of ammonia, much more sensitive than measuring NADPH based ammonia assay.

2. 
   

   Kit Contents:

   
   

Components	100 assays	Cap Color	Part Number
Ammonia Assay Buffer OxiRed Probe Dimethylsulfoxide (DMSO; Dried) Enzyme Mix (lyophilized) Converting Enzyme (Lyophilized) NH4Cl Standard (100 mM)	25 ml 1 vial 0.4 ml 1 vial 1 vial 100 µl	WM Red Brown Green Blue Yellow	K370-100-1 K370-100-2 K370-100-3 K370-100-4 K370-100-5 K370-100-6

3. 
   

   Storage and Handling:

   
   

Store the kit at -20^oC, protect from light. Allow Assay Buffer to warm to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay. All the solution in this kit should be kept capped when not in use to prevent absorption of ammonia from the air.

4. 
   

   Reagent preparation:

   
   

Probe: Dissolve in 220 l DMSO (provided) before use. Store at -20^oC, protect from light and moisture.

Enzyme Mix and Converting Enxyme: Dissolve in 220 l Assay Buffer separately. Aliquot to prevent multiple freeze/thaw cycle. Store at -20^oC. Use within two months.

5. 
   

   Ammonia Assay Protocol:

   
   

 

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 dH2O, 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 (2x10⁶) can be homogenized in 100 l Assay Buffer, centrifuge at 13,000 xg for 10 minutes to remove insoluble material. Liquid sample can be tested directly. Add 2-50 l 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.

 

 

 

 

 

Note: Pyruvate in samples will interfere with the assay. If significant amount of pyruvate is suspected in your sample, set a pyruvate 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 44 μl 46 μl

OxiRed Probe 2 μl 2 μl

Enzyme Mix 2 μl 2 μl

Converting Enzyme 2 l 0 μl

Add 50 μl of the Reaction Mix to each well containing the NH₄Cl Standard and test samples. Add 50 l Sample Control Mix to pyruvate 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 NH₄Cl from all readings (The background reading can be significant and must be subtracted from readings). Subtract the pyruvate Sample Control readings from sample readings. Plot NH₄Cl standard Curve, NH₄Cl 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.