ab83360

Ammonia Assay Kit

(Colorimetric)

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

Table of Contents
INTRODUCTION 1
1. BACKGROUND 1
2. ASSAY SUMMARY 2
GENERAL INFORMATION 3
3. PRECAUTIONS 3
4. STORAGE AND STABILITY 3
5. LIMITATIONS 4
6. MATERIALS SUPPLIED 4
7. MATERIALS REQUIRED, NOT SUPPLIED 5
8. TECHNICAL HINTS 6
ASSAY PREPARATION 7
9. REAGENT PREPARATION 7
10. STANDARD PREPARATION 8
11. SAMPLE PREPARATION 9
ASSAY PROCEDURE 11
12. ASSAY PROCEDURE 11
DATA ANALYSIS 13
13. CALCULATIONS 13
14. TYPICAL DATA 14
RESOURCES 17
15. QUICK ASSAY PROCEDURE 17
16. TROUBLESHOOTING 18
17. INTERFERENCES 20
18. FAQS 20
19. NOTES 21
 INTRODUCTION

INTRODUCTION

1. BACKGROUND
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 (max = 570 nm) which can be easily quantified by plate reader. The
kit can detect 1 nmol (~20 M) 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.

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 INTRODUCTION

2. ASSAY SUMMARY

Sample preparation

Standard curve preparation

Add reaction mix and incubate at 37C for 60 minutes

Measure optical density (OD570 nm)

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 GENERAL INFORMATION

GENERAL INFORMATION

3. PRECAUTIONS
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.

4. STORAGE AND STABILITY

Store kit at -20C 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.

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 GENERAL INFORMATION

5. LIMITATIONS
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.

6. MATERIALS SUPPLIED
Item Amount Storage Storage
Condition Condition
(Before (After
Preparation) Preparation)
Ammonia Assay Buffer 25 mL -20C -20C
OxiRed Probe 200 L -20C -20C
Developer 1 vial -20C -20C
Enzyme Mix 1 vial -20C -20C
(Lyophilized)
Converting Enzyme 1 vial -20C -20C
(Lyophilized)
NH4Cl Standard (10 mM) 100 L -20C -20C

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 GENERAL INFORMATION

7. MATERIALS REQUIRED, NOT SUPPLIED

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 (ddH2O)
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)

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 GENERAL INFORMATION

8. TECHNICAL HINTS
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.

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 ASSAY PREPARATION

ASSAY PREPARATION

9. REAGENT 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 -20C.
9.2. OxiRed Probe:
Ready to use as supplied. Warm by placing in a 37C bath for 1- 5
minutes to thaw the DMSO solution before use. NOTE: DMSO
tends to be solid when stored at -20C, even when left at room
temperature, so it needs to melt for few minutes at 37C. Store at
- 20C protected from light. Once the probe is thawed, use with
two months.
9.3. Developer:
Reconstitute in 220 L Ammonia Assay Buffer. Aliquot developer
so that you have enough volume to perform the desired number of
assays Store at -20C. Once reconstituted, use within 2 months.
9.4. Enzyme Mix:
Reconstitute in 220 L Ammonia Assay Buffer. Aliquot enzyme so
that you have enough volume to perform the desired number of
assays. Store at -20C. Once reconstituted, use within 2 months.
9.5. Converting Enzyme:
Reconstitute in 220 L Ammonia Assay Buffer. Aliquot converting
enzyme so that you have enough volume to perform the desired
number of assays. Store at -20C. 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
-20C. Keep on ice while in use.

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 ASSAY PREPARATION

10. STANDARD 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 L of 1 mM Ammonium Chloride Standard by adding
10 L of the 10 mM Ammonium Chloride Standard to 90 L of
ddH2O.
10.2. Using 1 mM standard, prepare standard curve dilution as
described in the table in a microplate or microcentrifuge tubes:
Standard # Volume of Assay Final End Conc
Standard Buffer volume ammonia in
(L) (L) standard in well
well (L) (nmol/well)
1 0 150 50 0
2 6 144 50 2
3 12 138 50 4
4 18 132 50 6
5 24 126 50 8
6 30 120 50 10

Each dilution has enough amount of standard to set up duplicate

readings (2 x 50 L).

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 ASSAY PREPARATION

11. SAMPLE 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
-80C. 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 106 cells equivalent of 1-5 x 104
cells/well required).
11.1.2. Wash cells in cold PBS.
11.1.3. Resuspend cells in 100 L 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 4C 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 g/well
required).
11.2.2. Wash tissue in cold PBS.
11.2.3. Resuspend tissue in 100 L Assay Buffer.

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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 4C 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 4C
during preparation.
11.3.2. Remove cells by centrifugating sample 10 minutes at 1,000
x g at 4C.
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 L/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 L/well of plasma; < 0.5
L of urine.

NOTE: We suggest using different volumes of sample to ensure

readings are within the Standard Curve range.

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 ASSAY PROCEDURE

ASSAY PROCEDURE

12. 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 L standard dilutions
Sample wells = 2-50 L samples (adjust volume to 50 L/well with
Assay Buffer).
Sample Background Control wells = 2 50 L samples (adjust
volume to 50 L/well with Assay Buffer).
12.2. Ammonia Reaction Mix
12.2.1. Prepare 50 L 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 L component x (Number reactions +1).

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 ASSAY PROCEDURE

Component Reaction Mix (L) Background

Reaction Mix (L)
Ammonia Assay Buffer 42 44
OxiRed Probe 2 2
Enzyme Mix 2 2
Developer 2 2
Converting Enzyme 2 0

12.3. Add 50 L of Reaction Mix into each standard and sample wells.
12.4. Add 50 L Sample Control Reaction Mix to Sample Control wells.
12.5. Mix and incubate at 37C for 60 minutes protected from light.
12.6. Measure output immediately on a colorimetric microplate reader at
OD 570 nm.

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 DATA ANALYSIS

DATA ANALYSIS

13. CALCULATIONS
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.
For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
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:
= ( )

Where:
Sa = amount of ammonia in the sample well calculated from
standard curve (nmol).
Sv = sample volume added into the wells (L).
D = Sample dilution factor.

NH4+ Molecular Weight = 18.04 g/mol.

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 DATA ANALYSIS

14. TYPICAL DATA

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).

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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).

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 DATA ANALYSIS

Figure 5: Ammonia measured in biological fluids, background signal subtracted

(duplicates +/- SD).

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 RESOURCES

RESOURCES

15. QUICK ASSAY PROCEDURE

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 L), samples (50 L) and
sample background control wells (50 L).
Prepare a master mix for Ammonia Reaction Mix and (if appropriate)
a master mix for Background Reaction Mix:
Component Reaction Mix Background
(L) Reaction Mix
(L)
Ammonia Assay Buffer 42 44
OxiRed Probe 2 2
Enzyme Mix 2 2
Developer 2 2
Converting Enzyme 2 0

Add 50 L Reaction Mix to standard and sample wells.

Add 50 L Background Reaction Mix to the sample background
control wells.
Incubate at 37C for 60 mins protected from light.
Measure plate immediately at OD 570 nm in a microplate reader.

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 RESOURCES

16. TROUBLESHOOTING
Problem Cause Solution
Buffers must be at room
Use of ice-cold buffer
temperature
Assay not Plate read at Check the wavelength and filter
incorrect wavelength settings of instrument
working
Colorimetric: Clear plates
Use of a different 96-
Fluorometric: black wells/clear
well plate
bottom plate
Samples not
Use provided protocol for
deproteinized (if
deproteinization
indicated on protocol)
Cells/tissue samples
Use Dounce homogenizer,
not homogenized
increase number of strokes
completely
Sample with Samples used after
Aliquot and freeze samples if
erratic multiple free/ thaw
needed to use multiple times
readings cycles
Use of old or Use fresh samples or store at -
inappropriately stored 80C (after snap freeze in liquid
samples nitrogen) till use
Presence of
Check protocol for interfering
interfering substance
substances; deproteinize samples
in the sample
Improperly thawed Thaw all components completely
Lower/ components and mix gently before use
Higher Allowing reagents to
Always thaw and prepare fresh
readings in sit for extended times
reaction mix before use
samples and on ice
Standards Incorrect incubation Verify correct incubation times
times or temperatures and temperatures in protocol

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 RESOURCES

Problem Cause Solution

Pipetting errors in Avoid pipetting small volumes
standard or reaction (< 5 L) and prepare a master mix
Standard mix whenever possible
readings do Air bubbles formed in Pipette gently against the wall of
not follow a well the tubes
linear pattern Standard stock is at
Always refer to dilutions described
incorrect
in the protocol
concentration
Measured at incorrect
Check equipment and filter setting
wavelength
Samples contain
Troubleshoot if it interferes with
Unanticipated interfering
the kit
results substances
Sample readings
Concentrate/ Dilute sample so it is
above/ below the
within the linear range
linear range

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 RESOURCES

17. INTERFERENCES
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.

18. FAQs
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 L 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.

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 RESOURCES

19. NOTES

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