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。
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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。
产品介绍
铁是人体必需的元素之一，对人体的新陈代谢和身体健康具有重要作用，血液中铁的含量不足，就会造成缺铁性贫血。铁存在于人体所有的细胞内，各组织、器官包括各内分泌腺都含有铁。肝脏、脾脏和肺组织内含量较为丰富。成人体内含铁3～5克，大部分都以蛋白质复合物形式存在，极少部分以离子的形态存在。本试剂盒可用于动植物组织和血清 (浆)或其他液体样本中的亚铁离子定量。样本中的Fe²⁺在酸性条件下和亚铁嗪反应生成紫红色化合物，在562nm处有吸收峰，通过测定该波长吸光度即可计算Fe²⁺的含量。
| F1515878 | 组分 | 物理外观 | 48T | 储存 | 单次测试使用量 |
| F1515878A | 缓冲液 | 液体 | 45 mL | 2-8℃. 避光 | 按需 |
| F1515878B | 显色液 | 液体 | 21 mL | 2-8℃. 避光 | 300 μL |
| F1515878C | 亚铁离子标准品 (40mM) | 液体 | 0.5 mL | 2-8℃. 避光 | 10 μL |
| F1515878D | 标准品稀释液 | 液体 | 15 mL | 2-8℃. 避光 | 按需 |
| F1515878E | 提取液 | 液体 | 45 mL | 2-8℃. 避光 | 按需 |
使用方法
1. 样品处理
(1) 组织样本：按照每0.01 g组织加入0.1 mL提取液的比例，用匀浆器或研钵冰浴匀浆。12000× g，4℃离心 10 min，收集上清液，置冰上待测。
(2) 血清 (浆)等液体样本：澄清的液体样本可直接检测。如果样本浑浊，5000× g，4℃离心 5 min，取上清液，置冰上待测。
注：本试剂盒提取液不能使用 BCA 蛋白定量，如需测定蛋白浓度，推荐使用阿拉丁即用型Bradford蛋白定量试剂盒 (去垢剂兼容型) (B1509656)。
2. 样品稀释
(1) 为确保样品数值在标准曲线范围内，建议进行预实验将样品设置多个稀释倍数，以确定样品中亚铁离子的大致浓度，如果数值不在标准曲线范围内，请调整样品的稀释倍数。
(2) 样本如需要稀释，血清 (浆)等液体样本的稀释建议使用缓冲液，而组织样本的稀释建议使用提取液。
3. 绘制标准曲线
将40 mmol/L (40000 μmol/L)亚铁离子标准品用标准品稀释液进行稀释得到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) | 标准品稀释液体积 (μL) | 稀释后浓度 (μmol/L) |
| 1 | 40000 | 10 | 990 | 400 |
| 2 | 400 | 400 | 1200 | 100 |
| 3 | 100 | 800 | 800 | 50 |
| 4 | 50 | 800 | 800 | 25 |
| 5 | 25 | 800 | 800 | 12.5 |
| 6 | 12.5 | 800 | 800 | 6.25 |
| 7 | 6.25 | 800 | 800 | 3.125 |
备注：下述实验中每个标准品管需600 μL标准品（注意不要在此步骤直接检测吸光度）。
4. 样品测定
(1) 可见光分光光度计预热 30 min 以上，调节波长到 562 nm。
(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 | 空白对照管 | 标准品管 | 测定样品管 |
| 样品 (μL) | 0 | 0 | 600 |
| 标准品 (μL) | 0 | 600 | 0 |
| 缓冲液 (μL) | 600 | 0 | 0 |
| 显色液 (μL) | 300 | 300 | 300 |
(3) 混匀，37°C 孵育30 min，若浑浊则需12000× g，离心 10 min。取800 μL上清液至1mL玻璃比色皿 (光径1cm)中。以空白管调零，于波长562nm处读取标准品管和测定样品管的吸光度A。标准品管吸光度记为A标准，测定样品管吸光度记为A测定。
注：本试剂盒建议使用1mL玻璃比色皿测量吸光度。若比色皿容量大于1mL，可将反应体系各组分加样量等比例放大以满足检测需求。
5. 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，A标准 为 y 轴，绘制标准曲线，得到标准方程，将 A测定 带入方程得到样品浓度Cₛₐₘₚₗₑ (μmol/L)。
5.2 亚铁离子浓度计算
5.2.1 血清（浆）等液体样本：亚铁离子含量 (μmol/L) = Cₛₐₘₚₗₑ × f；
5.2.2 组织样本：
(1) 以组织湿重进行计算： 亚铁离子含量 (μmol/kg wet weight) = Cₛₐₘₚₗₑ × f ÷ (m/V)；
(2) 按蛋白浓度计算：亚铁离子含量 (nmol/mg 蛋白) = Cₛₐₘₚₗₑ × f ÷ Cₚᵣ
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)；
Cₚᵣ：样本蛋白质浓度，mg/mL。
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。亚铁离子易被氧化，样本放置时间较长或反复冻融会造成结果不准确。
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：
Product Description
Iron is one of the essential elements for the human body and plays a vital role in metabolism and overall health. Insufficient iron in the blood can lead to iron deficiency anemia. Iron is present in all cells of the human body, and various tissues and organs, including endocrine glands, contain iron. The liver, spleen, and lung tissues are relatively rich in iron. The adult human body contains 3–5 grams of iron, most of which exists in the form of protein complexes, with only a small fraction in ionic form. This kit is intended for the quantitative determination of ferrous ions in animal and plant tissues, serum (plasma), and other liquid samples. In an acidic medium, Fe²⁺ reacts with ferrozine to produce a purple‑red complex that exhibits a maximum absorbance at 562 nm. The concentration of Fe²⁺ is then determined by measuring the absorbance at this wavelength.
| F1515878 | Components | Appearance | 48T | Storage | Quantity Per Test |
| F1515878A | Buffer Solution | Liquid | 45 mL | 2-8℃. Store in the dark | As needed |
| F1515878B | Chromogenic Solution | Liquid | 21 mL | 2-8℃. Store in the dark | 300 μL |
| F1515878C | Ferrous Ion Standard (40mM) | Liquid | 0.5 mL | 2-8℃. Store in the dark | 10 μL |
| F1515878D | Standard Diluent | Liquid | 15 mL | 2-8℃. Store in the dark | As needed |
| F1515878E | Extraction Buffer | Liquid | 45 mL | 2-8℃. Store in the dark | As needed |
Protocol
1. Sample Preparation
(1) Tissue samples: Add 0.1 mL of Extraction Buffer per 0.01 g of tissue, and homogenize using a homogenizer or mortar and pestle on ice. Centrifuge at 12,000× g and 4 °C for 10 minutes, collect the supernatant, and keep it on ice until analysis.
(2) Serum, plasma, and other liquid samples: Clear liquid samples can be tested directly. If the sample is turbid, centrifuge at 5000× g and 4 °C for 5 minutes, collect the supernatant, and keep it on ice until analysis.
Note: The Extraction Buffer provided in this kit is not compatible with BCA protein assays. If protein concentration determination is required, it is recommended to use Aladdin’s Ready‑to‑Use Bradford Protein Assay Kit (Detergent Compatible) (B1509656).
2. Sample Dilution
(1) To ensure that the sample values fall within the standard curve range, it is recommended to perform a preliminary experiment with multiple dilution factors to determine the approximate concentration of ferrous ions in the sample. If the values are not within the standard curve range, adjust the dilution factor accordingly.
(2) If dilution is necessary, use Buffer Solution for serum/plasma and other liquid samples, and use Extraction Buffer for tissue samples.
3. Preparation of Standard Curve
Dilute the 40 mmol/L (40,000 μmol/L) Ferrous Ion Standard with Standard Diluent to obtain standards at concentrations of 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) | Standard Diluent Volume (μL) | Final Conc. (μmol/L) |
| 1 | 40000 | 10 | 990 | 400 |
| 2 | 400 | 400 | 1200 | 100 |
| 3 | 100 | 800 | 800 | 50 |
| 4 | 50 | 800 | 800 | 25 |
| 5 | 25 | 800 | 800 | 12.5 |
| 6 | 12.5 | 800 | 800 | 6.25 |
| 7 | 6.25 | 800 | 800 | 3.125 |
Note: Each standard tube requires 600 μL of diluted standard for the subsequent assay (do not measure absorbance at this step).
4. Sample Measurement
(1) Pre‑warm the visible spectrophotometer for at least 30 minutes. Set the wavelength to 562 nm.
(2) Prepare the reaction mixtures as shown below. Include a blank control (no sample).
| Reagent | Blank | Standard | Sample |
| Sample (μL) | 0 | 0 | 600 |
| Standard (μL) | 0 | 600 | 0 |
| Buffer Solution (μL) | 600 | 0 | 0 |
| Chromogenic Solution (μL) | 300 | 300 | 300 |
(3) After thorough mixing, incubate the mixture at 37 °C for 30 minutes. If the solution becomes turbid, centrifuge at 12,000 × g for 10 minutes. Transfer 800 μL of the supernatant to a 1‑mL glass cuvette (with a 1‑cm light path). Zero the instrument using the blank tube, and then read the absorbance (A) of the standard tube and the sample tube at 562 nm. Record the standard absorbance as Astandard, and the sample absorbance as Aₛₐₘₚₗₑ.
Note: A 1‑mL glass cuvette is recommended for absorbance readings. For cuvettes larger than 1 mL, proportionally increase all reagent volumes accordingly.
5. Calculation of ferrous ion content
5.1 Preparation of the standard curve: Plot the standard concentration (x‑axis) against Astandard(y‑axis) to obtain the linear equation. Substitute Aₛₐₘₚₗₑ into the equation to calculate the sample concentration Cₛₐₘₚₗₑ (μmol/L).
5.2 Calculation of Fe²⁺ concentration
5.2.1 For serum/plasma or other liquid samples: Fe²⁺ content (μmol/L) = Cₛₐₘₚₗₑ × f
5.2.2 For tissue samples:
(1) Based on tissue wet weight: Fe²⁺ content (μmol/kg wet weight) = Cₛₐₘₚₗₑ × f ÷ (m / V)
(2) Based on protein concentration: Fe²⁺ content (nmol/mg protein) = Cₛₐₘₚₗₑ × f ÷ Cₚᵣ
Note:
f: dilution factor before measurement;
m: tissue wet weight (g);
V: volume of Extraction Buffer added (mL);
Cₚᵣ: protein concentration of the sample (mg/mL).
Precautions:
1. During the procedure, avoid contact of samples and reagents with iron‑containing materials to prevent contamination.
2. Fresh samples are recommended. Fe²⁺ is easily oxidized; prolonged storage or repeated freeze‑thaw cycles may compromise accuracy.
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:
_%E7%94%BB%E6%9D%BF%201.jpg)
| F1515878 | 组分 | 物理外观 | 48T | 储存 | 单次测试使用量 |
| F1515878A | 缓冲液 | 液体 | 45 mL | 2-8℃. 避光 | 按需 |
| F1515878B | 显色液 | 液体 | 21 mL | 2-8℃. 避光 | 300 μL |
| F1515878C | 亚铁离子标准品 (40mM) | 液体 | 0.5 mL | 2-8℃. 避光 | 10 μL |
| F1515878D | 标准品稀释液 | 液体 | 15 mL | 2-8℃. 避光 | 按需 |
| F1515878E | 提取液 | 液体 | 45 mL | 2-8℃. 避光 | 按需 |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F1039887 |
分析证书 | F1515878 |
危险品化学品经营许可证（带存储）