Urease (UE) Activity Assay Kit (IPB, Micro Method)

Cat. No.: U1521990

GRADE & PURITY BioReagent

概要

Urease (UE, EC 3.5.1.5) catalyzes the hydrolysis of urea to produce ammonia and carbonic acid. UE activity is positively correlated with organic matter content, total nitrogen and available nitrogen levels, serving as an indicator of soil and tissue nitrogen status.

This kit adopts indophenol blue colorimetry to quantify NH₃-N generated from urea hydrolysis by urease, with maximum absorbance at 630 nm. 

Item No.	Appearance	Components	48T	96T	Storage
U1521990A	Liquid	Extraction Solution	70 mL	70 mL×2	2-8 ℃
U1521990B	Powder	Reagent Ⅰ	1 vial	1 vial	2-8 ℃
U1521990C	Liquid	Reagent Ⅱ	25 mL	50 mL	2-8 ℃
U1521990D	Liquid	Reagent Ⅲ A	0.4 mL	0.8 mL	2-8 ℃
U1521990E	Liquid	Reagent Ⅲ B	1.6 mL	3.2 mL	2-8 ℃
U1521990F	Liquid	Reagent Ⅳ	2 mL	4 mL	2-8 ℃. Store in the dark.
U1521990G	Liquid	Standard	0.2 mL	0.4 mL	2-8 ℃

Notes：

1.Verify the volume of each kit component before starting experiments.

2.All reagents are supplied with an extra 10% volume on top of the standard specification for standard curve preparation and preliminary trial tests.

Self-supplied Instruments and Reagents

Microplate reader, transparent 96-well microplates, PBS, deionized water, tissue homogenizer (for tissue samples), constant temperature incubator, refrigerated centrifuge, adjustable pipettes and pipette tips

General Precautions

1.Conduct preliminary trials with 2–3 samples exhibiting expected large differences before formal detection.

2.For tissue, cell and bacterial samples, normalize final results by total protein concentration to eliminate inter-sample variation.

3.This kit is compatible with spectrophotometer detection; proportionally adjust reagent volumes following spectrophotometer operating requirements.

4.It is recommended to generate an independent standard curve for each experiment to improve detection accuracy. If no self-made standard curve is available, calculate results using the typical standard curve formula provided in the Results section.

5.Biochemical assay reagents are generally irritant and biohazardous. Wear full personal protective equipment including lab coat, mask, gloves and hairnet throughout all operations, and perform all experimental steps in a fume hood or biosafety cabinet for occupational safety.

6.This product is for research use only; not intended for clinical diagnostic applications.

Experimental Procedures

1. Reagent Preparation

Reagent Name	Preparation Instructions	Notes & Precautions
Extraction Solution	Ready-to-use; equilibrate to room temperature before use	Store at 2-8 ℃
Working Reagent Ⅰ	Prepare immediately before use; for 48 T, add 9 mL deionized water; for 96 T, add 18 mL deionized water, fully dissolve before use	Store at 2-8 ℃
Reagent Ⅱ	Ready-to-use; equilibrate to room temperature before use	Store at 2-8 ℃
Working Reagent Ⅲ	Prepare immediately before use; pour Solution A into Solution B and mix well before use	Stable for 1 week when stored at 4℃; toxic and with pungent odor, perform all operations in a fume hood
Reagent Ⅳ	Ready-to-use; equilibrate to room temperature before use	Store at 4℃, protected from light
Standard	1 M standard solution, ready-to-use; equilibrate to room temperature before use	Store at 2-8℃

2. Standard Working Solution Preparation

Dilute 1 M NH₄Cl Standard with Extraction Solution to prepare a 1 mM stock standard. Further serially dilute the 1 mM standard solution as specified in the table below. Prepare and analyze a standard curve alongside every independent experiment. Diluted standard solutions are unstable and must be used within 4 hours of preparation.

No.	Standard (μL)	Extraction Solution (μL)	Concentration (µM)
1	200 µL of 1 mM stock	0	1000
2	100 µL of 1000 μM standard	100	500
3	100 µL of 500 μM standard	100	250
4	100 µL of 250 μM standard	100	125
5	100 µL of 125 μM standard	100	62.5
6	100 µL of 62.5 μM standard	100	31.25
7	100 µL of 31.25 μM standard	100	15.625
Blank	0	100	0

3. Sample Preparation

Notes: Fresh samples are recommended for immediate detection. If testing cannot be performed right away, samples may be stored at −80 °C for up to 1 month.

Animal & Plant Tissues: Weigh approximately 0.1 g tissue sample, add 1 mL Extraction Solution, homogenize on ice, centrifuge at 8000 g, 4 °C for 10 min. Collect the supernatant and keep on ice prior to assay.

Cells or Bacteria: Harvest 5×10⁶ cells/bacteria, wash with cold PBS, centrifuge at 800 g for 2 min and discard supernatant. Resuspend pellet in 1 mL Extraction Solution, lyse by sonication on ice for 5 min (20% power / 200 W, 3 s sonication / 7 s interval, repeat 30 cycles). Centrifuge lysate at 8000 g, 4 °C for 10 min, collect supernatant and store on ice.

Serum / Plasma: Detect directly without pre-processing.

4. Assay Protocol

(1) Microplate reader setup: Preheat the instrument for ≥30 min and set detection wavelength to 630 nm.

(2) Enzymatic reaction: Prepare reaction mixtures in 1.5 mL EP tubes as outlined below:

Reagent	Assay Tube (μL)	Control Tube (μL)
Sample	20	20
Working Reagent Ⅰ	90	0
Deionized Water	0	90
Reagent Ⅱ	190	190

(3) Thoroughly mix all tubes, incubate in a 37 °C water bath for 1 h, then centrifuge at 10000 g, 25 °C for 10 min. Collect supernatant and dilute 10-fold (mix 0.1 mL supernatant with 0.9 mL deionized water).

(4) Ammonia quantification: Pipette reagents into a 96-well plate following the plate layout below (blank and standard wells only require single measurement per run).

Reagent	Test Well (μL)	Control Well (μL)	Standard Well (μL)	Blank Well (μL)
Diluted Supernatant	80	80	0	0
Standard Solution	0	0	80	0
Deionized Water	0	0	0	80
Working Reagent Ⅲ	15	15	15	15
Reagent Ⅳ	15	15	15	15
Deionized Water	90	90	90	90

Notes: Mix thoroughly after adding Reagent Ⅳ, then incubate at room temperature for 20 min.

(5)  Absorbance measurement: Mix each well rapidly, read absorbance at 630 nm, record values asA_sample, A_control, A_standard and A_blank respectively.

Calculation of Urease Activity

Two equivalent formulas are provided below: the full derivation formula and simplified computational formula.

1 Raw Data Calculation

2 Standard Curve Construction

ΔA_sample = A_sample − A_control 

ΔA_standard = A_standard − A_blank 

Plot standard curve with standard concentration as the y-axis and ΔA_standard as the x-axis. Substitute ΔA_sample into the curve equation to obtain y value (unit: μM).

3 Urease Activity Calculation

● Calculation by Tissue Weight: 

Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH₃-N per gram of tissue per minute. 

UE (U/g)=y×(V_{total-reaction}÷V_sample)×10×V_extract÷T÷1000÷W=0.0025y÷W 

● Calculation by Total Protein Concentration: 

Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH₃-N per milligram of total protein per minute. 

UE (U/mg prot)=y×(V_{total-reaction}÷V_sample)×10÷T÷1000÷Cpr=0.0025y÷Cpr 

●Calculation by Cell/Bacterial Count::

Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH-N per 10⁶ cells or bacteria per minute. 

UE (U/10⁶)=y×(V_{total-reaction}÷V_sample)×10×V_extract÷T÷1000÷N=0.0025y÷N 

●Calculation by Serum/Plasma Volume:

Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH₃-N per milliliter of serum/plasma per minute. T: Enzymatic reaction incubation time = 60 min 

UE (U/mL)=y×(V_{total-reaction}÷V_sample)×10÷T÷1000=0.0025y

V_{total-reaction}: Total volume of enzymatic reaction system = 0.3 mL

V_sample: Volume of sample added to reaction = 0.02 mL

1000: Unit conversion factor (1 L = 1000 mL)

Cpr: Total protein concentration of sample (mg/mL)

10: 10-fold dilution factor for post-reaction supernatant

V_extraction: Volume of Extraction Solution used for sample lysis = 1 mL

W: Weight of tissue sample (g)

N: Cell/bacterial count in units of 10⁶ (e.g., 5×10⁶ cells corresponds to N=5)

Representative Results

Typical Standard Curve:

Calculation Examples:

1.Tissue sample example: 0.1 g mouse liver tissue was assayed in 96-well plate. 

ΔA_sample = 0.206 − 0.09 = 0.116. Substitute into standard curve to get y = 196.995. UE (U/g) = 0.0025 × 196.995 ÷ 0.1 = 4.924 U/g

2.Cell sample example: 1×10⁶ Jurkat cells were assayed.

 ΔA_sample = 0.110 − 0.044 = 0.066. Substitute into standard curve to get y = 111.7753, N=5. UE (U/10⁶ cells) = 0.0025 × 111.7753 ÷ 5 = 0.056 U/10⁶

3.Serum example: 20μL bovine serum was assayed. 

ΔA_sample = 0.210 − 0.093 = 0.117. Substitute into standard curve to get y = 198.689. UE (U/mL serum) = 0.0025 × 198.689 = 0.497 U/mL 

FAQ

What to do if measured ΔA_sample  is excessively high or low?

If ΔA_sample > 0.6: Dilute the original sample with Extraction Solution or reduce the starting sample weight/cell number, then re-assay.

If ΔA_sample < 0.001: Increase the mass/volume of starting sample appropriately before re-testing.

Specifications

同義語

Microvolume Assay Kit for Urease (UE) Activity

仕様と純度

BioReagent

等級

BioReagent

アプリケーション

Enzyme activity assay

サンプルタイプ

プラズマ, 動物組織, 植物組織, 細菌, 血清

検出方法

IPB, Spectrophotometry

定量的

Quantitative

Detection instrument

Microplate reader

Detection wavelength

630nm

感度

Light-sensitive

保管と配送

保管条件

Store at 2-8°C,Protected from light

入荷

Wet ice

安定性と保管

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

Item No.	Appearance	Components	48T	96T	Storage
U1521990A	Liquid	Extraction Solution	70 mL	70 mL×2	2-8 ℃
U1521990B	Powder	Reagent Ⅰ	1 vial	1 vial	2-8 ℃
U1521990C	Liquid	Reagent Ⅱ	25 mL	50 mL	2-8 ℃
U1521990D	Liquid	Reagent Ⅲ A	0.4 mL	0.8 mL	2-8 ℃
U1521990E	Liquid	Reagent Ⅲ B	1.6 mL	3.2 mL	2-8 ℃
U1521990F	Liquid	Reagent Ⅳ	2 mL	4 mL	2-8 ℃. Store in the dark.
U1521990G	Liquid	Standard	0.2 mL	0.4 mL	2-8 ℃