概述规格储存与运输技术文档高级数据质检证书(CoA,COO,BSE/TSE 和分析图谱) 溶液计算器

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文献证明

在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。

概述

铁是人体必需的微量元素之一，它是血红蛋白、肌红蛋白、细胞色素及其他酶系统的主要成分，帮助氧的运输，促进脂肪氧化。缺乏铁元素容易造成贫血、代谢紊乱，并影响机体的免疫功能。在酸性条件下，铁从复合物中解离出来，然后使用还原剂将Fe³⁺ 还原为 Fe²⁺，使 Fe²⁺ 在酸性条件下与三吡啶基三嗪（TPTZ）形成蓝色配合物。该配合物在 593 nm 处有吸收峰，测定该波长下的吸光度即可检测总铁含量。若不对 Fe³⁺ 进行还原处理，则可检测亚铁离子含量。本试剂盒可用于动植物组织、细胞和血清（浆）或其他液体样本中的总铁和亚铁离子定量。

T1529721	组分	物理外观	48T	96T	储存	单次测试使用量
T1529721A	检测缓冲液	液体	15 mL	30 mL	2-8℃	按需
T1529721B	还原缓冲液	液体	6 mL	12 mL	2-8℃	100 μL
T1529721C	显色液	液体	6 mL	12 mL	2-8℃. 避光	100 μL
T1529721D	铁标准品 (40mM)	液体	0.5 mL	1 mL	2-8℃. 避光	10 μL
T1529721E	提取液	液体	15 mL	30 mL	2-8℃	按需

使用方法

1. 样品处理

1.1 动植物组织：称取约 0.1 g 样本，加入 1 mL 提取液，室温匀浆，12000×g，室温离心 10 min，取上清液待测。

1.2 细胞：收集 5×10⁶ 个细胞到离心管内，加入 1 mL 提取液，超声波破碎细胞 5 min（功率200W，超声 3 s，间隔 7 s，重复 30 次），12000×g，室温离心 10 min，取上清液待测。

1.3 血清（浆）等液体样本：澄清液体样本可直接检测，若样本浑浊，12000×g，室温离心 10 min，取上清液待测。

注：对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁的BCA蛋白定量试剂盒（货号：B665595）。本试剂盒提取液不能用于蛋白含量测定，如需测定蛋白含量，用去离子水重新提取样本后进行蛋白浓度测定。

2. 样品稀释

2.1 细胞和血清样本的铁离子和亚铁离子含量较低，通常不建议稀释样品。动物组织匀浆上清建议用提取液稀释 4 倍后检测，结果乘以稀释倍数。

2.2 样本如需要稀释，血清（浆）等液体样本的稀释建议使用检测缓冲液，而细胞和组织样品的稀释建议使用提取液。

3. 绘制标准曲线：将40 mmol/L（40000 μmol/L）铁标准品用检测缓冲液进行稀释得到400、100、50、25、12.5、6.25、3.125 μmol/L的标准品，现配现用。标准品稀释可参考下表：

序号	稀释前浓度（μmol/L）	标准品体积（μL）	检测缓冲液体积（μL）	稀释后浓度（μmol/L）
1	40000	10	990	400
2	400	100	300	100
3	100	150	150	50
4	50	150	150	25
5	25	150	150	12.5
6	12.5	150	150	6.25
7	6.25	150	150	3.125

备注：下述实验中每个标准管需100 μL标准品。

4. 样品测定

4.1 酶标仪预热 30 min 以上，调节波长到 593 nm。

4.2 按下表设置反应体系加样。设置不加样品的空白对照管。

试剂名称	空白管	标准品管	亚铁离子测定样品管	总铁离子测定样品管
样品 (μL)	0	0	100	100
标准品 (μL)	0	100	0	0
还原缓冲液 (μL)	0	100	0	100
检测缓冲液 (μL)	200	0	100	0
显色剂 (μL)	100	100	100	100

4.3 混匀，37°C 孵育30 min，反应后液体澄清可直接取200 μL上清液于酶标板各对应孔中。若浑浊则需12000× g，室温离心 5 min后取200 μL上清液于酶标板各对应孔中，在酶标仪593 nm处测定各孔OD值。空白孔的OD值记为A_空白，标准品孔的OD值记为A_标准，亚铁离子测定样品孔的OD值记为A_Fe²⁺。总铁离子测定样品孔的OD值记为A_Total。计算：ΔA_Fe²⁺= A_Fe²⁺- A_空白，ΔA_Total= A_Total- A_空白, Δ A_标准 = A_标准 - A_空白。

5. 总铁离子和亚铁离子含量计算

5.1 标准曲线的绘制：以标准品浓度为x轴，Δ A_标准为 y 轴，绘制标准曲线，得到标准方程，将 ΔA_Fe²⁺或者 ΔA_Total 代入标准曲线，即可计算出亚铁离子浓度，记录为B_Fe²⁺和总铁离子浓度，记录为B_Total。

5.2 根据实验需求选择适当的公式计算铁离子浓度C。

5.2.1 血清（浆）等液体样本：C (μmol/L) = B × f；

5.2.2 组织样本：

(1) 以组织湿重进行计算：C (μmol/kg wet weight) = B × f ÷ (m/V)；

(2) 按蛋白浓度计算：C (nmol/mg protein) = B × f ÷ C_pr

5.2.3 细胞样本：

(1) 以细胞数量进行计算： C (nmol/10⁶ cells ) = B × f ÷ (N/V)

(2) 按蛋白浓度计算：C (nmol/mg protein) = B × f ÷ C_pr

注：

f：样本加入检测体系前的稀释倍数；

m：组织湿重质量（g）；

V：样本提取液加入量(mL)；

C_pr：样本蛋白质浓度，mg/mL；

N: 用于裂解的细胞样本数量/10⁶ 。

注意事项

1. 操作过程中，样品与试剂等避免与铁器接触，以防污染。

2. 建议使用新鲜样本。如果不立即进行实验，样本可在-80℃保存 1 个月。

3. 所测样本的值高于标准曲线的上限，应稀释样品后重新测定。

4. 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。

5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。

结果展示

典型标准曲线：

Iron is one of the essential trace elements for the human body. It is a major component of hemoglobin, myoglobin, cytochromes, and other enzyme systems, helping to transport oxygen and promote fat oxidation. Iron deficiency can easily lead to anemia and metabolic disorders, and can affect the body's immune function. Under acidic conditions, iron dissociates from complexes. A reducing agent is then used to reduce Fe³⁺ to Fe²⁺, allowing Fe²⁺ to form a blue complex with tripyridyltriazine (TPTZ) under acidic conditions. This complex exhibits an absorption peak at 593 nm, and the total iron content can be calculated by measuring the absorbance at this wavelength. If Fe³⁺ is not reduced, the ferrous ion content can be detected instead. This kit can be used for the quantification of total iron and ferrous ions in animal and plant tissues, cells, serum (plasma), or other liquid samples.

T1529721	Component	Appearance	48T	96T	Storage	Quantity Per Test
T1529721A	Assay Buffer	Liquid	15 mL	30 mL	2-8℃	As needed
T1529721B	Reduction Buffer	Liquid	6 mL	12 mL	2-8℃	100 μL
T1529721C	Chromogenic Solution	Liquid	6 mL	12 mL	2-8℃. Store in the dark	100 μL
T1529721D	Iron Standard (40mM)	Liquid	0.5 mL	1 mL	2-8℃. Store in the dark	10 μL
T1529721E	Extraction Buffer	Liquid	15 mL	30 mL	2-8℃	As needed

Protocol

1. Sample Preparation

1.1 Animal and plant tissues: Weigh approximately 0.1 g of sample, add 1 mL of Extraction Buffer, and homogenize at room temperature. Centrifuge at 12,000 × g for 10 min at room temperature, and collect the supernatant for testing.

1.2 Cells: Collect 5 × 10⁶ cells into a centrifuge tube, add 1 mL of Extraction Buffer, and sonicate the cells for 5 min (power 200 W, sonication 3 s, interval 7 s, 30 cycles). Centrifuge at 12,000 × g for 10 min at room temperature, and collect the supernatant for testing.

1.3 Liquid samples such as serum (plasma): Clear liquid samples can be tested directly. If the sample is turbid, centrifuge at 12,000 × g for 10 min at room temperature, and collect the supernatant for testing.

Note: For tissue and cell samples, results can be normalized between samples by determining protein concentration. Aladdin’s BCA Protein Assay Kit (Cat. No. B665595) is recommended. The Extraction Buffer in this kit cannot be used for protein content determination. If protein content determination is required, re‑extract the sample with deionized water before measuring protein concentration.

2. Sample Dilution

2.1 The total iron ion content in cell and serum samples is relatively low, so dilution is generally not recommended. It is recommended to dilute the animal tissue homogenate supernatant 4‑fold with Extraction Buffer prior to detection, and multiply the result by the dilution factor.

2.2 If sample dilution is required, serum (plasma) and other liquid samples are recommended to be diluted with Assay Buffer , while cell and tissue samples are recommended to be diluted with Extraction Buffer.

3. Preparation of the Standard Curve: Dilute the 40 mmol/L (40,000 μmol/L) iron standard with Assay Buffer to obtain standards at 400, 100, 50, 25, 12.5, 6.25, and 3.125 μmol/L. Prepare fresh immediately before use. Refer to the table below for standard dilution.

No.	Pre‑dilution Conc. (μmol/L)	Std. Volume (μL)	Assay Buffer Volume (μL)	Final Conc. (μmol/L)
1	40000	10	990	400
2	400	100	300	100
3	100	150	150	50
4	50	150	150	25
5	25	150	150	12.5
6	12.5	150	150	6.25
7	6.25	150	150	3.125

Note: Each standard tube requires 100 μL of diluted standard for the subsequent assay.

4. Sample Measurement

4.1 Pre‑warm the microplate reader for at least 30 min. Set the wavelength to 593 nm.

4.2 Prepare the reaction mixtures as shown below. Include a blank control (no sample).

Reagent	Blank Tube	Standard Tube	Fe²⁺ Sample Tube	Total Iron Sample Tube
Sample (μL)	0	0	100	100
Standard (μL)	0	100	0	0
Reduction Buffer (μL)	0	100	0	100
Assay Buffer (μL)	200	0	100	0
Chromogenic Solution (μL)	100	100	100	100

4.3 Mix well and incubate at 37°C for 30 min. If the liquid is clear after the reaction, directly transfer 200 μL of the supernatant to the corresponding wells of the microplate. If turbid, centrifuge at 12,000 × g for 5 min at room temperature, then transfer 200 μL of the supernatant to the corresponding wells of the microplate, and measure the OD value of each well at 593 nm using a microplate reader. Record the OD value of the blank well as A_blank, the OD value of the standard well as A_standard, the OD value of the ferrous ion sample well as A_Fe²⁺, and the OD value of the total iron sample well as A_Total. Calculate: ΔA_Fe²⁺ = A_Fe²⁺ − A_blank; ΔA_Total = A_Total − A_blank; ΔA_standard = A_standard − A_blank.

5. Calculation of Total Iron and Ferrous Ion Content

5.1 Preparation of the standard curve: Plot the standard curve using the standard concentrations as the x-axis and the corresponding ΔA_standard values as the y-axis to obtain the standard equation. Substitute ΔA_Fe²⁺ or ΔA_Total into the standard curve to calculate the ferrous ion concentration (recorded as B_Fe²⁺) and the total iron ion concentration (recorded as B_Total), respectively.

5.2 Select the appropriate formula according to experimental requirements to calculate the iron ion concentration C.

5.2.1 For liquid samples such as serum (plasma): C (μmol/L) = B × f

5.2.2 For tissue samples:

(1) Calculated based on tissue wet weight: C (μmol/kg wet weight) = B × f ÷ (m/V)

(2) Calculated based on protein concentration: C (nmol/mg protein) = B × f ÷ C_pr

5.2.3 For cell samples:

(1) Calculated based on cell number: C (nmol/10⁶ cells) = B × f ÷ (N/V)

(2) Calculated based on protein concentration: C (nmol/mg protein) = B × f ÷ C_pr

Notes:

f: dilution factor of the sample before addition to the assay system;

m: wet weight of the tissue (g);

V: volume of Extraction Buffer added to the sample (mL);

C_pr: protein concentration of the sample (mg/mL);

N: number of cells used for lysis (in units of 10⁶).

Precautions

1. During the procedure, avoid contact of samples and reagents with iron-containing materials to prevent contamination.

2. Fresh samples are recommended. If the assay is not performed immediately, samples can be stored at –80 °C for up to 1 month.

3. If the measured value of a sample exceeds the upper limit of the standard curve, the sample should be diluted and re-assayed.

4. Before the formal assay, it is recommended to perform a preliminary experiment using 2–3 samples that are expected to show significant differences.

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

Results Presentation

Typical standard curve:

规格

规格或纯度

BioReagent, 标曲法

英文名称

Total Iron and Ferrous Ion Content Assay Kit (TPTZ, Micro Method)

应用

细胞代谢

检测范围

0.78-400 μM

检测方法

分光光度法

定量&半定量

定量

检测仪器

酶标仪

检测波长

593 nm

敏感性

Light-sensitive

检测时长

70 min

样本体积

100μL

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

各组分在相应的储存条件下保质期一年。

组分与储存

T1529721	组分	物理外观	48T	96T	储存	单次测试使用量
T1529721A	检测缓冲液	液体	15 mL	30 mL	2-8℃	按需
T1529721B	还原缓冲液	液体	6 mL	12 mL	2-8℃	100 μL
T1529721C	显色液	液体	6 mL	12 mL	2-8℃. 避光	100 μL
T1529721D	铁标准品	液体	0.5 mL	1 mL	2-8℃. 避光	10 μL
T1529721E	提取液	液体	15 mL	30 mL	2-8℃	按需

技术文档

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溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

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浓度

体积

分子量

g/mol

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计算配制储备溶液所需的稀释度。

浓度1（C1）

体积 1 (V1)

浓度 2 (C2)

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复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

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所需的复溶浓度

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总铁与亚铁离子含量检测试剂盒 (TPTZ, 微量法)

储存与运输

质量文档

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概述

规格

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✅ 分析证书 (COA)

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