植物铵态氮含量检测试剂盒 (靛酚蓝, 微量法)

货号: P1527535
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级别和纯度: BioReagent ? 生物试剂级(BioReagent)—— 经测试适用于生命科学和分子生物学。适用于需要生物相容性的细胞培养、检测和生化工作。 标曲法 ?
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48T
P1527535-48T
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¥849.90
96T
P1527535-96T
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¥1,399.90
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BioReagent,标曲法 级 ,适用于对基线干扰要求严格的色谱和分析工作流程。

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概述

铵态氮可以被植物直接吸收利用,也可经过硝化微生物的作用转化为硝态氮,进而被植物或微生物同化为 有机氮化物。 本试剂盒检测原理是铵态氮在强碱介质中能够与次氯酸钠和苯酚反应,生成水溶性蓝色染料靛酚蓝,产物 在 625 nm 处有特征吸收峰,吸光值与铵态氮含量成正比。 

组分表

组分货号组分名称物理外观48T96T储存
P1527535A提取液液体70 mL70 mL×22-8℃
P1527535B试剂 I固体1 vial2 vials2-8℃,避光保存
P1527535C试剂 II液体6 mL12 mL2-8℃,避光保存
P1527535D试剂 III液体1.5 mL3 mL2-8℃
P1527535E标准品 (500 µmol/mL)液体1 mL1 mL2-8℃

*请在实验前检查各组分的量。 

*各组分在规格基础上额外提供 10%的量用于进行标准曲线制作或预实验。 

自备仪器与试剂

资源类型资源名称备注
仪器酶标仪能测 625 nm 处的吸光度
耗材96 孔酶标板普通透明板
试剂去离子水配制试剂用
其他匀浆器(组织样本)、恒温箱、低温离心机、可调节式移液枪及枪头检测量较大时合理使用多通道移液器有助于提高实验效率

注意事项 

1. 正式检测前,建议选择 2-3 个预期差异较大的样本进行预实验。

2. 建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线,可参考结果展示部分的典型标 准曲线公式进行结果计算。 

3. 生化检测试剂普遍具有刺激性、生物毒性等,为了您的安全和健康,请在实验全程做好生物安全防护,穿 戴实验服、口罩、手套、头套等安全护具,在通风橱、生物安全柜中进行实验。 

4. 本产品仅供科学研究使用,不适用于临床诊断。 

试剂准备

试剂名称试剂准备注意事项
提取液即用型;使用前平衡到室温4℃保存
试剂 I临用前配制,根据实验用量取一支试剂 I加入6 mL去离子水充分溶解现配现用;有一定毒性,建议在通风橱进行实验
试剂 II即用型;使用前平衡到室温4℃避光保存
试剂 III即用型;使用前平衡到室温4℃保存
标准品 (500 µmol/mL)即用型;使用前平衡到室温4℃保存

标准品配制 

使用 500 μmol/mL 标准品,按照下表所示,进一步稀释标准品。每次实验都要做一次标准品检测,制作 标曲;稀释后的标准品溶液不稳定,必须在 4 小时内使用。 

序号标准品 (μL)提取液 (μL)浓度 (μmol/mL)
15 μL 500 μmol/mL9952.5
2100 μL of 2.5 μmol/mL1001.25
3100 μL of 1.25 μmol/mL1000.625
4100 μL of 0.625 μmol/mL1000.313
5100 μL of 0.313 μmol/mL1000.156
6100 μL of 0.156 μmol/mL1000.078
7100 μL of 0.078 μmol/mL1000.039
空白01000

样本制备

注意:推荐使用新鲜样本,如果不立即进行实验,样本可在-80℃保存 1 个月。

植物组织:称取 0.1 g 植物组织,加入 1 mL 提取液室温匀浆,然后 10000 g,4℃离心 10 min,取上清待测。

实验步骤

1. 酶标仪准备:预热 30 min 以上,调节波长到 625 nm。

2. 检测体系配制:在 96 孔板中按照如下方式操作

试剂空白孔(μL)标准孔(μL)测定孔(μL)
样本上清0020
提取液2000
标品0200
试剂 I 工作液808080
试剂 II
808080

充分混匀,25℃静置1h

试剂 III
202020

吸光度检测:充分混匀,立即测定 625 nm 处的吸光值,分别记为 A空白、A标准 和 A测定。 

数据处理 

1.计算 ΔA测定 = A测定 − A空白,ΔA标准 = A标准 − A空白。 

2.标准曲线绘制:以标准品浓度为 y 轴,ΔA标准 为 x 轴,绘制标准曲线。将 ΔA测定 代入方程得到 y 值(μmol/mL)。 

3.样本铵态氮含量计算: 铵态氮含量 (μg/g) = y × V提取 × 18 ÷ W × n = y × 18 ÷ W × n 

V提取:加入 Extraction Buffer 体积,1 mL;18:NH4+ 摩尔质量,μg/μmol;W:样品质量,g;n:样本稀释倍数。

结果展示 

典型标准曲线:

示例-1: 取 0.1 g 烟草茎,按照测定步骤操作,用 96 孔板进行检测。测得ΔA测定 = A测定 - A空白 =0.146-0.059=0.087, 代入标准曲线,计算 y=0.131。计算得:铵态氮含量(μg/g)=y×V 提取×18÷W×n=y×18÷W×n=23.58 μg/g。  

FAQ  

检测得到的样本ΔA测定 测定过高或者过低怎么办? 如果样本ΔA测定 >2.0,需用提取液适当进行稀释后,再进行实验,结果乘以稀释倍数,或减少提取 用样本量。如果样本的ΔA测定 <0.04,可适当提高样本量。 

Ammonium nitrogen can be directly absorbed and utilized by plants, or it can be converted to nitrate nitrogen through the action of nitrifying microorganisms, which is then assimilated by plants or microorganisms into organic nitrogen compounds. The detection principle of this kit is that ammonium nitrogen reacts with sodium hypochlorite and phenol in a strong alkaline medium to form a water-soluble blue dye, indophenol blue. The product has a characteristic absorption peak at 625 nm, and the absorbance value is directly proportional to the ammonium nitrogen content. 

Component Table 

Component No.Component NameAppearance48T96TStorage
P1527535AExtraction BufferLiquid70 mL70 mL×22-8℃
P1527535BReagent ISolid1 vial2 vials2-8℃, Store in the dark.
P1527535CReagent IILiquid6 mL12 mL2-8℃, Store in the dark.
P1527535DReagent IIILiquid1.5 mL3 mL2-8℃
P1527535EStandard (500 µmol/mL)Liquid1 mL1 mL2-8℃

*Please check the quantity of each component before the experiment.

*An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.

Reagents, consumables and Equipments not provided 

Resource TypeResource NameRemarks
InstrumentMicroplate readerCapable of measuring absorbance at 625 nm
Consumable96-well microplateStandard transparent plate
ReagentDeionized waterFor reagent preparation
OtherHomogenizer (for tissue samples), incubator, refrigerated centrifuge, adjustable pipettes and tipsUsing a multi-channel pipette can improve efficiency when processing large numbers of samples

Precautions

1.Before formal testing, it is recommended to select 2–3 samples with expected large differences for preliminary experiments.

2..It is recommended that users establish their own standard curve to improve result accuracy. If a standard curve is not prepared independently, the typical standard curve formula provided in the Results section may be used for calculation.

3.Biochemical assay reagents are generally irritant and biologically toxic. For your safety and health, please take appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, head covers, and other protective equipment. Conduct experiments in a fume hood or biosafety cabinet.

4.This product is for scientific research use only and is not intended for clinical diagnosis.

Reagent Preparation

Reagent NameReagent PreparationNotes
Extraction BufferReady-to-use; equilibrate to room temperature before useStore at 4°C
Reagent IPrepare immediately before use. Add 6 mL deionized water to one vial of Reagent I and dissolve thoroughly.Prepare fresh; toxic; prepare in a fume hood
Reagent IIReady-to-use; equilibrate to room temperature before useStore at 4°C, protected from light
Reagent IIIReady-to-use; equilibrate to room temperature before useStore at 4°C
Standard (500 µmol/mL)Ready-to-use; equilibrate to room temperature before useStore at 4°C

Standard Preparation

Use the 500 µmol/mL standard to prepare further dilutions as shown in the table below. A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

No.Standard (μL)Extraction Buffer (μL)Concentration (μmol/mL)
15 μL of 500 μmol/mL9952.5
2100 μL of 2.5 μmol/mL1001.25
3100 μL of 1.25 μmol/mL1000.625
4100 μL of 0.625 μmol/mL1000.313
5100 μL of 0.313 μmol/mL1000.156
6100 μL of 0.156 μmol/mL1000.078
7100 μL of 0.078 μmol/mL1000.039
Blank01000

Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.

Plant tissues: Weigh 0.1 g of plant tissue, add 1 mL of Extraction Buffer, and homogenize at room temperature. Centrifuge at 10,000 × g for 10 min at 4°C, then collect the supernatant for analysis.

Assay Procedure

1.Microplate reader preparation: Preheat for at least 30 min and set the wavelength to 625 nm.

2.Reaction system preparation: In a 96-well plate, proceed as follows:

ReagentBlank well (μL)Standard well (μL)Sample well (μL)
Sample supernatant0020
Extraction Buffer2000
Standard0200
Reagent I working solution808080
Reagent II808080
Mix thoroughly and incubate at 25°C for 1 h


Reagent III202020

Absorbance measurement: Mix thoroughly and measure the absorbance at 625 nm immediately. Record as Ablank, Astandard, and Asample, respectively. 

Data Processing 

1.Calculate ΔAsample = Asample − Ablank, and ΔAstandard = Astandard − Ablank. 

2.Standard curve construction: Plot the standard concentration on the y-axis against ΔAstandard on the x-axis. Substitute ΔAsample into the equation to obtain the y value (μmol/mL). 

3.Calculation of ammonium nitrogen content in samples: Ammonium nitrogen content (μg/g) = y × Vextraction × 18 ÷ W × n = y × 18 ÷ W × n 

Vextraction: Volume of Extraction Buffer added, 1 mL; 18: Molar mass of NH4+, μg/μmol; W: Sample weight, g; n: Sample dilution factor.

Results Display

Typical standard curve:

Example-1: Take 0.1 g of tobacco stem and follow the assay procedure using a 96-well plate. Measured ΔAsample = Asample − Ablank = 0.146 − 0.059 = 0.087. Substitute into the standard curve to obtain y = 0.131. Calculation: Ammonium nitrogen content (μg/g) = y × Vextraction × 18 ÷ W × n = y × 18 ÷ W × n = 23.58 μg/g. 

FAQ 

What if the measured ΔAsample is too high or too low? 

If ΔAsample > 2.0, dilute the sample appropriately with Extraction Buffer and repeat the assay, then multiply the result by the dilution factor, or reduce the amount of sample used for extraction. 

If ΔAsample < 0.04, increase the sample amount appropriately.

规格

别名
植物 NH₄⁺‑N 含量检测试剂盒 (微量靛酚蓝法)
英文别名
Ammonium‑Nitrogen / NH₄⁺‑N Assay Kit
规格或纯度
BioReagent, 标曲法
英文名称
Plant Ammonium Nitrogen Content Assay Kit (Indophenol Blue, Micro Method)
应用
氮代谢
检测范围
0.039-2.5 μmol/mL
检测方法
靛酚蓝, 分光光度法
检测仪器
酶标仪
检测波长
625nm
储存与运输
储存条件
2-8°C储存,避光
运输条件
低温运输
稳定性与储存
Store at 2-8℃ long term (12 months).Store in the dark.
组分与储存
组分货号组分名称物理外观48T96T储存
P1527535A提取液液体70 mL70 mL×22-8℃
P1527535B试剂 I固体1 vial2 vials2-8℃,避光保存
P1527535C试剂 II液体6 mL12 mL2-8℃,避光保存
P1527535D试剂 III液体1.5 mL3 mL2-8℃
P1527535E标准品 (500 µmol/mL)液体1 mL1 mL2-8℃

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