Instructions for use:
For the rapid, sensitive and accurate measurement of ammonia levels in various samples.
This product is for research use only and is not intended for diagnostic use.
Version 6 Last Updated: 9 December 2015

INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
LIMITATIONS
6.
MATERIALS SUPPLIED
7.
MATERIALS REQUIRED, NOT SUPPLIED
8.
TECHNICAL HINTS
ASSAY PREPARATION
9.
REAGENT PREPARATION
10.
STANDARD PREPARATION
11.
SAMPLE PREPARATION
ASSAY PROCEDURE
12.
ASSAY PROCEDURE
DATA ANALYSIS
13.
CALCULATIONS
14.
TYPICAL DATA
RESOURCES
15.
QUICK ASSAY PROCEDURE
16.
TROUBLESHOOTING
17.
INTERFERENCES
18.
FAQS
19.
NOTES
11
13
13
14
17
8
9
11
6
7
7
17
18
20
20
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4
5
3
3
3
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INTRODUCTION
INTRODUCTION
Ammonia Assay Kit (Colorimetric) (ab83360) provides a rapid, simple, sensitive, and reliable assay suitable for research and high throughput assay of Ammonia or Ammonium. In the assay, ammonia or ammonium 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 or ammonium, much more sensitive than measuring NADPH based ammonia assay.
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. ab83360 Ammonia Assay Kit (Colorimetric) 1

INTRODUCTION
Sample preparation
Standard curve preparation
Add reaction mix and incubate at 37°C for 60 minutes
Measure optical density (OD570 nm) ab83360 Ammonia Assay Kit (Colorimetric) 2

GENERAL INFORMATION
GENERAL INFORMATION
Please read these instructions carefully prior to beginning the assay.
 All kit components have been formulated and quality control tested to function successfully as a kit.
 We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances.
However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet.
 Reagents should be treated as possible mutagens and should be handle with care and disposed of properly. Please review the Safety
Datasheet (SDS) provided with the product for information on the specific components.
 Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas.
 All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures.
Store kit at -20ºC in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted.
Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Materials Supplied section.
Aliquot components in working volumes before storing at the recommended temperature. Reconstituted components are stable for
2 months.
All the solutions in this kit should be kept capped when not in use to prevent absorption of ammonia from the air.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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GENERAL INFORMATION
 Assay kit intended for research use only. Not for use in diagnostic procedures.
 Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted.
Item Amount
Ammonia Assay Buffer
OxiRed Probe
Developer
Enzyme Mix
(Lyophilized)
Converting Enzyme
(Lyophilized)
NH
4
Cl Standard (10 mM)
25 mL
200 µL
1 vial
1 vial
1 vial
100 µL
Storage
Condition
(Before
Preparation)
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
Storage
Condition
(After
Preparation)
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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GENERAL INFORMATION
These materials are not included in the kit, but will be required to successfully perform this assay:
 Microplate reader capable of measuring absorbance at OD 570 nm
 MilliQ water or other type of double distilled water (ddH
2
O)
 Pipettes and pipette tips, including multi-channel pipette
 Assorted glassware for the preparation of reagents and buffer solutions
 Orbital shaker
 Tubes for the preparation of reagents and buffer solutions
 96 well plate with clear flat bottom
 Dounce homogenizer (if using tissue) ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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GENERAL INFORMATION
 This kit is sold based on number of tests. A ‘test’ simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions.
 Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures.
 Avoid foaming or bubbles when mixing or reconstituting components.
 Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.
 Ensure plates are properly sealed or covered during incubation steps.
 Ensure all reagents and solutions are at the appropriate temperature before starting the assay.
 Samples which generate values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer.
 Make sure you have the right type of plate for your detection method of choice.
 Make sure all necessary equipment is switched on and set at the appropriate temperature.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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ASSAY PREPARATION
ASSAY PREPARATION
 Briefly centrifuge small vials at low speed prior to opening
9.1.
Ammonia Assay Buffer:
Ready to use as supplied. Equilibrate to room temperature before use. Store at -20°C.
9.2.
OxiRed Probe:
Ready to use as supplied. Warm by placing in a 37°C bath for 1- 5 minutes to thaw the DMSO solution before use. NOTE: DMSO tends to be solid when stored at -20°C, even when left at room temperature, so it needs to melt for few minutes at 37°C . Store at
- 20°C protected from light. Once the probe is thawed, use with two months.
9.3.
Developer:
Reconstitute in 220 µL Ammonia Assay Buffer. Aliquot developer so that you have enough volume to perform the desired number of assays Store at -20°C. Once reconstituted, use within 2 months.
9.4.
Enzyme Mix:
Reconstitute in 220 µL Ammonia Assay Buffer. Aliquot enzyme so that you have enough volume to perform the desired number of assays. Store at -20°C. Once reconstituted, use within 2 months.
9.5.
Converting Enzyme:
Reconstitute in 220 µL Ammonia Assay Buffer. Aliquot converting enzyme so that you have enough volume to perform the desired number of assays. Store at -20°C. Once reconstituted, use within
2 months.
9.6.
NH4Cl Standard:
Ready to use as supplied. Aliquot standard so that you have enough volume to perform the desired number of assays. Store at
-20°C. Keep on ice while in use.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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ASSAY PREPARATION
 Always prepare a fresh set of standards for every use.
 Discard the working standard dilutions after use as they do not store well.
10.1. Prepare 100 µL of 1 mM Ammonium Chloride Standard by adding
10 µL of the 10 mM Ammonium Chloride Standard to 90 µL of ddH
2
O.
10.2. Using 1 mM standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes:
Standard #
1
Volume of
Standard
(µL)
0
Assay
Buffer
(µL)
150
Final volume standard in well (µL)
50
End Conc ammonia in well
(nmol/well)
0
4
5
2
3
6
6
12
18
24
30
144
138
132
126
120
50
50
50
50
50
6
8
2
4
10
Each dilution has enough amount of standard to set up duplicate readings (2 x 50 µL).
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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ASSAY PREPARATION
General Sample Information
 We recommend performing several dilutions of your sample to ensure the readings are within the standard value range.
 We recommend that you use fresh samples. If you cannot perform the assay at the same time, we suggest that you complete the
Sample Preparation step before storing the samples. Alternatively, if that is not possible, we suggest that you snap freeze samples in liquid nitrogen upon extraction and store the samples immediately at
-80°C. When you are ready to test your samples, thaw them on ice.
Be aware however that this might affect the stability of your samples and the readings can be lower than expected.
11.1.
Cell (adherent or suspension) samples:
11.1.1. Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10 6 cells – equivalent of 1-5 x 10 4 cells/well required).
11.1.2. Wash cells in cold PBS.
11.1.3. Resuspend cells in 100 µL Assay Buffer.
11.1.4. Homogenize cells quickly by pipetting up and down a few times.
11.1.5. Centrifuge sample for 2 – 5 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material.
11.1.6. Collect supernatant and transfer to a clean tube.
11.1.7. Keep on ice.
11.2.
Tissue samples:
11.2.1. Harvest the amount of tissue necessary for each assay (initial recommendation = 10 mg – equivalent of 20-50 µg/well required).
11.2.2. Wash tissue in cold PBS.
11.2.3. Resuspend tissue in 100 µL Assay Buffer.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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ASSAY PREPARATION
11.2.4. Homogenise tissue with a Dounce homogenizer sitting on ice, with 10 – 15 passes.
11.2.5. Centrifuge samples for 2 – 5 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material.
11.2.6. Collect supernatant and transfer to a clean tube.
11.2.7. Keep on ice.
11.3.
Plasma:
11.3.1. Collect whole blood into heparin tubes. Keep sample at 4°C during preparation.
11.3.2. Remove cells by centrifugating sample 10 minutes at 1,000 x g at 4°C.
11.3.3. Collect supernatant and transfer to a clean tube. After centrifugation, it is important to immediately transfer into a clean tube.
11.3.4. Keep on ice
Initial sample recommendation = 5 – 15 µL/well of plasma.
11.4.
Urine and other biological fluids:
Urine and other biological fluids can be tested directly in the assay.
Initial sample recommendation = 5 – 15 µL/well of plasma; < 0.5
µL of urine.
NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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ASSAY PROCEDURE
ASSAY PROCEDURE
 Equilibrate all materials and prepared reagents to correct temperature prior to use.
 We recommended to assay all standards, controls and samples in duplicate.
 Prepare all reagents, working standards, and samples as directed in the previous sections.
 This product is very sensitive and reagents can react with other sources of ammonia present in laboratory. Ensure you keep the plate close with a lid when not pipetting, and work on a glovebox or a negative air pressure area if possible.
NOTE: Pyruvate in samples will interfere with the assay. If a significant amount of pyruvate is suspected in your samples, set up Sample
Controls. The pyruvate reading must be subtracted from sample readings.
12.1.
Set up Reaction Wells
Standard wells = 50 µL standard dilutions
Sample wells = 2-50 µL samples (adjust volume to 50 µL/well with
Assay Buffer).
Sample Background Control wells = 2 – 50 µL samples (adjust volume to 50 µL/well with Assay Buffer).
12.2.
Ammonia Reaction Mix
12.2.1. Prepare 50 µL of Reaction Mix for each reaction. Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master mix of the
Reaction Mix to ensure consistency. We recommend the following calculation:
X µL component x (Number reactions +1).
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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ASSAY PROCEDURE
Component
Ammonia Assay Buffer
OxiRed Probe
Enzyme Mix
Developer
Converting Enzyme
Reaction Mix (µL)
42
2
2
2
2
Background
Reaction Mix (µL)
44
2
2
2
0
12.3. Add 50 µL of Reaction Mix into each standard and sample wells.
12.4. Add 50 µL Sample Control Reaction Mix to Sample Control wells.
12.5. Mix and incubate at 37°C for 60 minutes protected from light.
12.6. Measure output immediately on a colorimetric microplate reader at
OD 570 nm.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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DATA ANALYSIS
DATA ANALYSIS
 Samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalyzed, then multiply the concentration found by the appropriate dilution factor.

13.1. Average the duplicate reading for each standard and sample.
13.2. Subtract the mean absorbance value of the blank (Standard #1) from all standard and sample readings. This is the corrected absorbance.
13.3. Plot the corrected absorbance values for each standard as a function of the final concentration of ammonia.
13.4. Draw the best smooth curve through these points to construct the standard curve. Most plate reader software or Excel can plot these values and curve fit. Calculate the trendline equation based on your standard curve data (use the equation that provides the most accurate fit).
13.5. Concentration of samples in the test samples is calculated as:
𝐴𝑚𝑚𝑜𝑛𝑖𝑎 𝑜𝑟 𝑎𝑚𝑚𝑜𝑛𝑖𝑢𝑚 =
( 𝑆𝑎
𝑆𝑣
)
∗ 𝐷
Where:
Sa = amount of ammonia in the sample well calculated from standard curve (nmol).
Sv = sample volume added into the wells (µL).
D = Sample dilution factor.
NH
4
+ Molecular Weight = 18.04 g/mol.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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DATA ANALYSIS
TYPICAL STANDARD CURVE – Data provided for demonstration purposes only . A new standard curve must be generated for each assay performed
Fig ure 1 : Typical ammonium standard calibration curve using colorimetric reading.
Figure 2: Ammonia measured in cell culture medium and control medium, background signal subtracted (duplicates +/- SD).
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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DATA ANALYSIS
Figure 3 : Ammonia measured in mouse tissue lysates (mg of extracted protein), background signal subtracted (duplicates +/- SD).
Figure 4: Ammonia measured in cell lysates, background signal subtracted
(duplicates +/- SD).
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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DATA ANALYSIS
Figure 5 : Ammonia measured in biological fluids, background signal subtracted
(duplicates +/- SD).
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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RESOURCES
RESOURCES
NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time.
 Solubilize enzyme mix, converting enzyme and developer, thaw
OxiRed probe, ammonia standard and ammonia Assay Buffer
(aliquot if necessary); get equipment ready
 Prepare Ammonia standard dilution [2 – 10 nmol/well]
 Prepare samples in optimal dilutions so that they fit standard curve readings.
 Set up plate in duplicate for standard (50 µL), samples (50 µL) and sample background control wells (50 µL).
 Prepare a master mix for Ammonia Reaction Mix and (if appropriate) a master mix for Background Reaction Mix:
Component Reaction Mix
(µL)
Ammonia Assay Buffer
OxiRed Probe
Enzyme Mix
Developer
Converting Enzyme
42
2
2
2
2
Background
Reaction Mix
(µL)
44
2
2
2
0
 Add 50 µL Reaction Mix to standard and sample wells.
 Add 50 µL Background Reaction Mix to the sample background control wells.
 Incubate at 37°C for 60 mins protected from light.
 Measure plate immediately at OD 570 nm in a microplate reader. ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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RESOURCES
Problem Cause
Assay not working
Sample with erratic readings
Lower/
Higher readings in samples and
Standards
Use of ice-cold buffer
Plate read at incorrect wavelength
Use of a different 96well plate
Samples not deproteinized (if indicated on protocol)
Cells/tissue samples not homogenized completely
Samples used after multiple free/ thaw cycles
Use of old or inappropriately stored samples
Presence of interfering substance in the sample
Improperly thawed components
Allowing reagents to sit for extended times on ice
Incorrect incubation times or temperatures
Solution
Buffers must be at room temperature
Check the wavelength and filter settings of instrument
Colorimetric: Clear plates
Fluorometric: black wells/clear bottom plate
Use provided protocol for deproteinization
Use Dounce homogenizer, increase number of strokes
Aliquot and freeze samples if needed to use multiple times
Use fresh samples or store at -
80°C (after snap freeze in liquid nitrogen) till use
Check protocol for interfering substances; deproteinize samples
Thaw all components completely and mix gently before use
Always thaw and prepare fresh reaction mix before use
Verify correct incubation times and temperatures in protocol ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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RESOURCES
Problem
Standard readings do not follow a linear pattern
Unanticipated results
Cause
Pipetting errors in standard or reaction mix
Air bubbles formed in well
Standard stock is at incorrect concentration
Measured at incorrect wavelength
Samples contain interfering substances
Sample readings above/ below the linear range
Solution
Avoid pipetting small volumes
(< 5 µL) and prepare a master mix whenever possible
Pipette gently against the wall of the tubes
Always refer to dilutions described in the protocol
Check equipment and filter setting
Troubleshoot if it interferes with the kit
Concentrate/ Dilute sample so it is within the linear range ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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RESOURCES
These chemicals or biological materials will cause interference in this assay causing compromised results or complete failure:
 Pyruvate – if significant amount of pyruvate is suspected in your
sample, set up a Sample control as described in Section 12.
What is the kit detecting: ammonium or ammonia?
This kit measures the total ammonia and total ammonium concentration in your samples.
Will media containing phenol red interfere with the assay reading?
Phenol red could interfere if the amount of medium used is enough to contribute to add red color to the sample. Typically, when diluted medium is used and the well volume is made up to 50 µL with buffer, phenol red will not interfere with the assay. However, since the probe emits at 570 nm and this is in the pink-orange-red zone of the spectrum, red color from the medium can interfere if undiluted medium is used.
ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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RESOURCES ab83360 Ammonia Assay Kit (Colorimetric) abXXXXXXX PRODUCT NAME
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