Enzymatic Method for Determining Urea (Phenylalanine Assay)

Introduction

This assay protocol is suitable for the colorimetric detection of urea in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Urea Assay Kit (MAK006). Urea concentration is determined by a coupled enzyme assay, which results in a colorimetric (570 nm) product, proportional to the Urea present. The linear range of detection for this assay is between 1.0–5.0 nmole.

Reagents

Urea Assay Buffer                                            25 mL
    Catalog Number MAK006A

Peroxide Substrate, in DMSO                           0.2 mL
    Catalog Number MAK006B

Enzyme Mix                                                    1 vl
    Catalog Number MAK006C

Developer                                                        1 vl
    Catalog Number MAK006D

Converting Enzyme                                          1 vl
    Catalog Number MAK006E

Urea Standard, 100 mM                                    0.1 mL
    Catalog Number MAK006F

Reagents and Equipment Required but Not Provided

96 well flat-bottom plate – it is recommended to use clear plates for colorimetric assays (Catalog Number M4436 or equivalent).

Spectrophotometric multiwell plate reader.

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Urea Assay Buffer – allow buffer to come to room temperature before use.

Peroxidase Substrate – Thaw at room temperature to melt solution prior to use. Aliquot and store protected from light and moisture at –20 °C.

Enzyme Mix, Developer, and Converting Enzyme – reconstitute each with 220 µL of Urea Assay Buffer. Mix well by pipetting, then aliquot and store, protected from light, at –20 °C. Use within 2 months of reconstitution and keep cold while in use.

Storage / Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate.

Urea Standards for Colorimetric Detection
Dilute 5 µL of the 100 mM (100 nmole/µL) Urea Standard Solution with 995 µL of Urea Assay Buffer to prepare a 0.5 mM (0.5 nmole/µL) standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the 0.5 mM Urea standard solution into a 96 well plate, generating 0 (blank), 1, 2, 3, 4, and 5 nmole/well standards. Add Urea Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation

Tissue (20 mg) or cells (2 x 106) should be rapidly homogenized in 100 µL of cold Urea Assay buffer. Centrifuge at 13,000 x g for 10 minutes at 4 °C to remove insoluble material.

Serum and other liquid samples can be directly added to the wells.

Bring samples to a final volume of 50 µL with Urea Assay Buffer.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

Ammonium ion, NAD+/NADP+, and pyruvate present in the sample can generate background. To control for background, include a blank sample for each sample by omitting the Converting Enzyme in the Reaction Mix.  

Assay Reaction

1.  Set up the Reaction Mixes according to the scheme in Table 1. 50 µL of the appropriate Reaction Mix is required for each reaction (well).

Table 1. Reaction Mixes

Reagent Sample Blank Samples and Standards
Urea Assay Buffer 44 µL 42 µL
Peroxidase Substrate 2 µL
2 µL
Enzyme Mix 2 µL 2 µL
Developer 2 µL 2 µL
Converting Enzyme -
2 µL

2.  Add 50 µL of the appropriate Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 60 minutes at 37 °C. Protect the plate from light during the incubation.

3.  Measure the absorbance at 570 nm (A570).

Results

Calculations

The background for the assays is the value obtained for the 0 (blank) Urea Standard. Correct for the background by subtracting the 0 (blank) value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate Urea standards to plot a standard curve.

Note: A new standard curve must be set up each time the assay is run.

Subtract the blank sample value from the sample reading to obtain the corrected measurement. Using the corrected measurement, the amount of Urea present in the sample may be determined from the standard curve.

Concentration of Urea

        Sa/Sv = C

Sa = Amount of phenylalanine in unknown sample (nmole) from standard curve
Sv = Sample volume (µL) added into the wells
C = Concentration of Urea in sample

Urea molecular weight: 60.07 g/mole.

Sample Calculation

Amount of Urea (Sa) = 4.84 nmole
    (from standard curve)
Sample volume (Sv) = 50 µL
Concentration of Urea in sample

4.84 nmole/50 µL = 0.0968 nmole/µL

0.0968 nmole/µL x 60.07 ng/nmole= 5.81 ng/µL

Materials

     

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.  

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