Urease Activity Assay Kit

Catalog Number MAK120

Storage Temperature 2–8 °C
TECHNICAL BULLETIN

Product Description Reagents and Equipment Required but Not

Urease is an enzyme that catalyzes the hydrolysis of Provided.
urea into carbon dioxide and ammonia. Urease is found • Spectrophotometric multiwell plate reader
in bacteria, yeast, and certain higher plant species. • Clear 96-well flat-bottom plate
Urease activity is found in environmental and fecal • 10 kDal Molecular Weight Cut-Off Filter (Catalog
samples. Number Z677108 or equivalent)

The Urease Activity Assay kit provides a simple and Precautions and Disclaimer
direct procedure for measuring urease activity in This product is for R&D use only, not for drug,
biological and environmental samples. In this assay, household, or other uses. Please consult the Material
urease catalyzes the hydrolysis of urea resulting in the Safety Data Sheet for information regarding hazards
production of ammonia. The ammonia is determined by and safe handling practices.
the Berthelot method resulting in a colorimetric product
measured at 670 nm, proportionate to the urease Storage/Stability
activity present in the sample. One unit of urease is the This kit is shipped at room temperature. Storage at
amount of enzyme that catalyzes the formation of 2–8 °C, protected from light, is recommended.
1.0 µmole of ammonia per minute at pH 7.0.
Procedure
Components Sample Preparation
The kit is sufficient for 100 assays in 96 well plates. Soil and other samples can be extracted in Assay
Buffer (10 mM sodium phosphate, pH 7.0) using any
Assay Buffer, pH 7.0 20 mL established methods.
Catalog Number MAK120A
Notes: The presence of ammonia in samples will result
Urea 1.5 mL in assay background. Ammonia in samples can be
Catalog Number MAK120B removed by dialysis or filtration with a 10 kDa Molecular
Weight Cut-Off Filter.
Ammonium Chloride, 50 mM 100 µL
Catalog Number MAK120C For unknown samples, it is suggested to test several
sample dilutions to ensure the readings are within the
Reagent A 12 mL linear range of the standard curve.
Catalog Number MAK120D
Sample Blank Preparation
Reagent B 6 mL Certain samples, such as soil samples, may contain low
Catalog Number MAK120E concentrations of ammonia. To correct for sample
ammonia, immediately prior to assay step 4, prepare a
sample blank by mixing the following reagents in the
indicated order: 100 µL of Reagent A, 90 µL of sample
extract, 10 µL of urea, and 50 µL of Reagent B.
Continue with assay as written.
2

Ammonium Chloride Standards Assay Reaction

Bring all components to room temperature before use. 1. Transfer 90 µL of the standards into separate wells
Dilute 5 µL of the 50 mM Ammonium Chloride Standard of a 96 well plate.
with 495 µL of Assay Buffer to create a 500 µM
Ammonium Chloride Standard Solution. Dilute the 2. Transfer 90 µL of samples into separate wells of
500 µM Ammonium Chloride Solution as indicated in the plate. Samples should be diluted in Assay
Table 1 to create the standard curve. Buffer. Transfer 90 µL of Assay Buffer into a
separate well to serve as an Assay Blank.
Table 1.
Ammonium Standards 3. Add 10 µL of Urea to each well. Incubate at room
temperature for 10 minutes. For samples with low
NH4Cl Final suspected urease activity, for example some
Number Standard Buffer Ammonia environmental samples), incubate reaction for
(500 µM ) Concentration 2–4 hours at either 30 °C or 37 °C.
1 100 µL 0 µL 500 µM Note: Certain samples, such as soil samples, may
2 80 µL 20 µL 400 µM contain low concentrations of ammonia. To correct
3 60 µL 40 µL 300 µM for sample ammonia, prepare a Sample blank by
4 40 µL 60 µL 200 µM mixing the following reagents in the indicated order:
100 µL of Reagent A, 90 µL of sample extract,
5 30 µL 70 µL 150 µM
10 µL of urea, and 50 µL of Reagent B.
6 20 µL 80 µL 100 µM
7 10 µL 90 µL 50 µM
4. Add 100 µL of Reagent A to each well to terminate
8 0 µL 100 µL 0 µM the Urease reaction, tap plate to mix, and then add
50 µL of Reagent B to each well. Tap plate to mix
again.

5. Incubate the reaction for 30 minutes protected from

light.

6. Measure the absorbance at 670 nm (A670).

These assays can be adapted for use in standard 1 mL

cuvettes. To adapt to cuvettes follow protocol as for
96 well plates but scale up volumes as follows: 360 µL
of sample, 40 µL of urea, 400 µL of Reagent A, and
200 µL of Reagent B.
 3

Results The amount of urease enzymatic activity in the sample

Calculations can be calculated using the equation below.
Use the values obtained from the appropriate
ammonium chloride standards to plot a standard curve Urease Activity (units/L) = (A670)sample – (A670)blank × n
and determine the slope using linear regression fitting. Slope × t
Note: A new standard curve must be set up each time
the assay is run. where:
n = dilution factor
t = incubation time (10 minutes for standard assay).
(A670)blank = value for Assay Blank (Assay Buffer alone)

Note: If Urease activity is higher than 25 units/L, dilute

sample in assay buffer, repeat assay, and multiply by
dilution factor.

One unit of urease is the amount of enzyme that

catalyzes the formation of 1.0 µmole of ammonia per
minute at pH 7.0.
4

Troubleshooting Guide
Problem Possible Cause Suggested Solution
Bring components to room temperature prior
Cold reagents
to starting assay
Assay not working Omission of step in procedure Refer and follow Technical Bulletin precisely
Plate reader at incorrect wavelength Check filter settings of instrument
Type of 96 well plate used For colorimetric assays, use clear plates
Use the Assay Buffer provided or refer to
Samples prepared in incompatible buffer
Technical Bulletin for instructions
Repeat the sample homogenization,
Cell/Tissue culture samples were
increasing the length and extent of
incompletely homogenized
homogenization step.
Samples with erratic
Samples used after multiple freeze-thaw Aliquot and freeze samples if needed to use
readings
cycles multiple times
Presence of interfering substance in the
If possible, dilute sample further
sample
Use of old or inappropriately stored Use fresh samples and store correctly until
samples use
Thaw all components completely and mix
Improperly thawed components
gently before use
Use of expired kit or improperly stored Check the expiration date and store the
Lower/higher reagents components appropriately
readings in samples Allowing the reagents to sit for extended
Prepare reagents fresh for each assay
and standards times on ice
Refer to Technical Bulletin and verify correct
Incorrect incubation times or temperatures
incubation times and temperatures
Incorrect volumes used Use calibrated pipettes and aliquot correctly
Samples measured at incorrect
Check the equipment and filter settings
Unanticipated results wavelength
Samples contain interfering substances If possible, dilute sample further

LS,MAM 11/13-1

2013 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other
countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their
particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at
[Link] and/or on the reverse side of the invoice or packing slip.