Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*

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Urea dose response in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices) measured with Amplite™ Colorimetric Urea Assay Kit.
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200 Tests 10058 $195


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Overview

Ex/Em (nm)650/None
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsAbsorbance microplate reader
Category Small Molecule Detection
Diagnostic Molecules
Related Cell Metabolism
Secondary Reagents
Urea is the final degradation product of protein and amino acid metabolism in animals. It is produced in liver, secreted by kidney and excreted through urine. The determination of urea is very useful test in clinical laboratory to monitor health status. The Blood Urea Nitrogen (BUN) test is a measure of the amount of nitrogen in the blood in the form of urea and is primarily used, along with the creatinine test to evaluate kidney function, helping diagnose kidney diseases. Our Amplite™ Colorimetric Urea Assay Kit provides a simple and sensitive colorimetric method for the quantitation of urea concentration in biological samples such as serum, plasma and urine, etc. The assay is based on an enzyme-coupled reaction of urea in the assay buffer, and finally produces a blue colored product. The intensity of color produced is proportional to the concentration of urea in the sample, which can be measured colorimetrically at 660-670 nm. This Amplite™ Colorimetric Urea Assay Kit provides a simple assay to detect as little as 10 µM urea in a 150 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare urea standards or test samples (50 µL)
  2. Add urea working solution (50 µL)
  3. Incubate at room temperature or 37°C for 30 - 60 min
  4. Add Assay Buffer II
  5. Read Absorbance at 665 nm

Important notes
Thaw all the kit components to room temperature before starting the experiment.

Key parameters
Instrument:Absorbance microplate reader
Absorbance:665 nm
Recommended plate:Clear bottom
Preparation of stock solutions
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Assay Enzyme Mix stock solution (100X):
Add 100 µL of ddH2O into the vial of Assay Enzyme Mix (Component A) to make 100X Assay Enzyme Mix stock solution.

Preparation of standard solution
Urea standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10058

Add 1 μL of 1.0 M Urea Standard (Component D) to 999 µL of DPBS to generate 1.0 mM standard urea solution (US7). Take 1.0 mM urea standard solution to perform 1:3 serial dilutions to get remaining urea standard solutions (US6 - US1).

Preparation of working solution

Add 50 μL of reconstituted Assay Enzyme Mix stock solution (100X) into 5 mL Assay Buffer I (Component B) to make urea working solution. Note: The urea working solution should be used promptly and kept from light. The assay sensitivity will be decreased with longer storage time. Fresh urea working solution is recommended for the best result.

Sample experimental protocol

Table 1. Layout of urea standards and test samples in a clear bottom 96-well microplate. US= Urea Standards (US1 - US7, 1 to 1000 µM), BL=Blank Control, TS=Test Samples. 

BL BL TS TS
US1 US1 ... ...
US2 US2 ... ...
US3 US3    
US4 US4    
US5 US5    
US6 US6    
US7 US7    

Table 2. Reagent composition for each well.

Well Volume Reagent
US1 - US7 50 µL Serial Dilutions (1 to 1000 µM)
BL 50 µL DPBS
TS 50 µL test sample
  1. Prepare urea standards (US), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of urea working solution to each well of urea standard, blank control, and test samples to make the total urea assay volume of 100 µL/well. For a 384-well plate, add 25 µL of urea working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction for 30 - 60 minutes at room temperature or 37°C, protected from light.

  4. Add 50 µL of Assay Buffer II (Component C) to each well so that the total assay volume is 150 µL/well. For a 384-well plate, add 25 µL Assay Buffer II (Component C) to each well, for a total assay volume of 75 µL/well.

  5. Incubate at room temperature for 10 - 15 minutes, and monitor the absorbance increase at 660 - 670 nm using an absorbance microplate reader. Note: The color turns to yellow after Assay Buffer II (Component C) is added, and the wells with urea standard or samples will show blue-green color after incubation. The intensity of the color will reach the maximum in 15 - 30 minutes, and is proportional to the concentration of urea. Note: The final color is stable for ~1 hour in room temperature and the color intensity will decrease with time.
Example data analysis and figures

The reading (Absorbance) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Urea samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. Urea dose response in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices) measured with Amplite™ Colorimetric Urea Assay Kit.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

A novel and sensitive resonance scattering assay for detection of urea in serum coupled urease catalytic reaction and NH4+ associated particle reaction
Authors: Liang A, Qin H, Zhou L, Zhang Y, Ouyang H, Wang P, Jiang Z.
Journal: Bioprocess Biosyst Eng (2011): 639

Dimethyl formamide-free, urea-NaCl fluorescence in situ hybridization assay for Staphylococcus aureus
Authors: Lawson TS, Connally RE, Vemulpad S, Piper JA.
Journal: Lett Appl Microbiol. (2011)

Evaluation of diuron (3-[3,4-dichlorophenyl]-1,1-dimethyl urea) in a two-stage mouse skin carcinogenesis assay
Authors: Ferrucio B, Franchi CA, Boldrin NF, de Oliveira ML, de Camargo JL.
Journal: Toxicol Pathol (2010): 756

Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers
Authors: Ngoka LC.
Journal: Proteome Sci (2008): 30

Immobilization of urease from pigeonpea (Cajanus cajan) on agar tablets and its application in urea assay
Authors: Mulagalapalli S, Kumar S, Kalathur RC, Kayastha AM.
Journal: Appl Biochem Biotechnol (2007): 291

Improvement of the decision efficiency of the accuracy profile by means of a desirability function for analytical methods validation. Application to a diacetyl-monoxime colorimetric assay used for the determination of urea in transdermal iontophoretic extracts
Authors: Rozet E, Wascotte V, Lecouturier N, Preat V, Dewe W, Boulanger B, Hubert P.
Journal: Anal Chim Acta (2007): 239

Enzymatic assay for determination of bicarbonate ion in plasma using urea amidolyase
Authors: Kimura S, Yamanishi H, Iyama S, Yamaguchi Y, Kanakura Y.
Journal: Clin Chim Acta (2003): 179

New enzymatic assay for serum urea nitrogen using urea amidolyase
Authors: Kimura S, Iyama S, Yamaguchi Y, Kanakura Y.
Journal: J Clin Lab Anal (2003): 52

Utilization of lacrimal urea assay in the monitoring of hemodialysis: conditions, limitations and lacrimal arginase characterization
Authors: Farkas A, Vamos R, Bajor T, Mullner N, Lazar A, Hraba A.
Journal: Exp Eye Res (2003): 183

Stool antigen assay (HpSA) is less reliable than urea breath test for post-treatment diagnosis of Helicobacter pylori infection
Authors: Bilardi C, Biagini R, Dulbecco P, Iiritano E, Gambaro C, Mele MR, Borro P, Tessieri L, Zentilin P, Mansi C, Vigneri S, Savarino V.
Journal: Aliment Pharmacol Ther (2002): 1733


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Additional Documents

 
Safety Data Sheet (SDS)


Certificate of Analysis