Electrophoresis | 电泳

📚 Electrophoresis | 电泳

Electrophoresis is a laboratory technique used to separate charged molecules, such as proteins, nucleic acids, and amino acids, based on their differential migration in an electric field. It is a cornerstone method in biochemistry and molecular biology for analysing complex mixtures, determining molecular weight, and assessing purity.

电泳是一种利用带电分子在电场中迁移速率差异来分离蛋白质、核酸和氨基酸等物质的实验室技术。它是生物化学与分子生物学中分析复杂混合物、测定分子量及评估纯度的基础方法。

1. Introduction to Electrophoresis | 电泳简介

Electrophoresis relies on the principle that charged particles move towards the electrode of opposite charge when an electric potential is applied. The migration velocity depends on the net charge, size, shape, and the properties of the medium through which the molecules travel.

电泳的原理是：施加电势时，带电粒子会向带相反电荷的电极移动。迁移速度取决于分子的净电荷、大小、形状以及其所穿行介质的性质。

The term ‘electrophoresis’ comes from the Greek words ‘electron’ (amber, associated with electricity) and ‘phoresis’ (to carry). It was first developed by Arne Tiselius in the 1930s for the separation of serum proteins, earning him the Nobel Prize in Chemistry in 1948.

“电泳”一词源自希腊语“electron”（琥珀，与电相关）和“phoresis”（携带）。该技术由 Arne Tiselius 于 20 世纪 30 年代首创，用于分离血清蛋白，他因此获得 1948 年诺贝尔化学奖。

2. Principle of Electrophoretic Separation | 电泳分离原理

When a molecule with a net charge q is placed in an electric field of strength E, it experiences an electrostatic force F = qE. As the molecule moves, it encounters frictional resistance from the medium, so it reaches a steady terminal velocity v where the electrical force is balanced by the frictional force. The mobility μ = v/E is thus proportional to the charge and inversely proportional to the Stokes radius of the molecule.

当一个净电荷为 q 的分子置于电场强度为 E 的电场中时，会受到静电力 F = qE。分子移动时会受到介质的摩擦阻力，因此会达到一个恒定的终末速度 v，此时电力与摩擦力平衡。迁移率 μ = v/E 因此与电荷成正比，与分子的 Stokes 半径成反比。

In free solution, mobility depends strongly on the charge-to-size ratio. However, in a sieving medium such as a gel, larger molecules are retarded more than smaller ones, allowing separation by molecular size even if the charge-to-mass ratio is constant.

在自由溶液中，迁移率主要取决于电荷与大小的比值。而在凝胶等筛分介质中，大分子比小分子受到更多阻碍，即使荷质比恒定，也能按分子大小实现分离。

3. Factors Affecting Migration Rate | 影响迁移速率的因素

Factor	Effect on Migration
Net charge	Higher net charge → greater mobility; at isoelectric point (pI), net charge = 0, migration stops.
Size (molecular weight)	Larger molecules experience more frictional drag and slower migration through gels.
Shape	Globular proteins migrate faster than fibrous proteins of the same mass; supercoiled DNA migrates differently from linear DNA.
Electric field strength	Higher voltage increases migration speed but may cause heating and band distortion.
Buffer pH and ionic strength	pH determines the degree of ionisation of analytes; ionic strength affects conductivity and resolution.
Gel concentration (%T)	Higher gel percentage → smaller pores → greater sieving effect, useful for smaller molecules.

Understanding these factors allows chemists to design an electrophoretic method that gives optimal resolution for a given mixture. For instance, the buffer pH is selected so that the analytes carry distinctive charges, while the gel concentration is chosen to match the size range of interest.

了解这些因素能让化学家设计出对特定混合物有最佳分辨率的电泳方法。例如，选择缓冲液 pH 值使分析物带有不同的电荷，同时选择与所关注分子大小范围相匹配的凝胶浓度。

4. Gel Electrophoresis: Agarose and Polyacrylamide | 凝胶电泳：琼脂糖与聚丙烯酰胺

Gels serve as both an anti‑convective medium and a molecular sieve. The two most common gels are agarose and polyacrylamide. Agarose gels, derived from seaweed, have relatively large pores and are ideal for separating DNA fragments (100 bp – 25 kb) and large proteins. Polyacrylamide gels, formed by crosslinking acrylamide with bisacrylamide, have smaller, controllable pore sizes, making them suitable for proteins and small nucleic acids.

凝胶既可用作抗对流的介质，又可用作分子筛。最常用的两种凝胶是琼脂糖和聚丙烯酰胺。琼脂糖凝胶来自海藻，孔径较大，适合分离 DNA 片段（100 bp – 25 kb）和大蛋白质。聚丙烯酰胺凝胶由丙烯酰胺与亚甲基双丙烯酰胺交联而成，孔径较小且可控，适用于蛋白质与小核酸。

Agarose gels are typically cast horizontally and run submerged in buffer (submarine gel), while polyacrylamide gels are cast vertically between glass plates. The choice of gel also depends on the required resolution and whether the sample will be recovered afterwards.

琼脂糖凝胶通常水平倒入并浸没在缓冲液中（水平式），而聚丙烯酰胺凝胶则是在玻璃板之间垂直倒入。凝胶的选择还取决于所需的分辨率以及后续是否需要回收样品。

5. SDS‑PAGE: Separating Proteins by Size | SDS‑PAGE：按大小分离蛋白质

SDS‑PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is the standard method for separating proteins by molecular weight. The anionic detergent SDS denatures proteins and coats them with a uniform negative charge, so the intrinsic charge differences are eliminated. The polypeptide chains are then separated solely based on length (molecular weight) as they migrate through a polyacrylamide gel.

SDS‑PAGE（十二烷基硫酸钠聚丙烯酰胺凝胶电泳）是按分子量分离蛋白质的标准方法。阴离子去垢剂 SDS 使蛋白质变性并为其包裹上一层均匀的负电荷，从而消除了原有的电荷差异。多肽链随后在聚丙烯酰胺凝胶中迁移，完全按长度（分子量）进行分离。

Before loading, proteins are boiled with SDS and a reducing agent (e.g., 2‑mercaptoethanol or DTT) to break disulfide bonds, ensuring complete denaturation. A tracking dye (bromophenol blue) is added to monitor the run. After electrophoresis, proteins are visualised by staining (e.g., Coomassie Brilliant Blue or silver stain).

上样前，蛋白质与 SDS 和还原剂（如 2‑巯基乙醇或 DTT）一同煮沸，以打断二硫键，确保完全变性。加入示踪染料（溴酚蓝）来监控电泳进程。电泳结束后，通过染色（如考马斯亮蓝或银染）使蛋白质可视化。

The log of the molecular weight of a protein is inversely proportional to its relative mobility (Rf) in a properly chosen pore size range. A calibration curve using standard proteins (markers) allows molecular weight estimation.

在适当孔径范围内，蛋白质分子量的对数与其相对迁移率（Rf）成反比。用标准蛋白（分子量标记）制作校正曲线，可估算未知蛋白的分子量。

6. Native PAGE and Protein Charge | 非变性PAGE与蛋白质电荷

In native PAGE, proteins are separated without denaturing agents. Migration depends on the protein’s intrinsic net charge, size, and shape. This technique preserves biological activity and quaternary structure, making it useful for studying protein complexes and enzyme activity. The pH of the gel and running buffer must be chosen so that the proteins of interest are negatively charged and migrate towards the anode.

在非变性 PAGE 中，蛋白质无需变性剂即可分离。迁移取决于蛋白质原有的净电荷、大小和形状。该技术能保留生物活性和四级结构，因此可用于研究蛋白质复合物和酶活性。所选凝胶和电泳缓冲液的 pH 值必须使目标蛋白带负电并向阳极迁移。

Because charge differences among proteins are maintained, two proteins of similar mass but different pI values can be resolved. However, the correlation between mobility and molecular weight is not linear, so native PAGE is not used for mass determination.

由于保留了蛋白质之间的电荷差异，质量相近但等电点不同的两种蛋白质可以得到分离。但迁移率与分子量并非线性关系，因此非变性 PAGE 不用于分子量测定。

7. Isoelectric Focusing (IEF) | 等电聚焦

Isoelectric focusing separates proteins according to their isoelectric points (pI) in a pH gradient. A mixture of carrier ampholytes (small, synthetic molecules with a range of pI values) is used to establish a stable pH gradient across a gel under an electric field. When a protein is placed in this gradient, it migrates until it reaches the pH where its net charge is zero — its pI — at which point it stops and forms a sharp band.

等电聚焦根据蛋白质的等电点（pI）在 pH 梯度中进行分离。使用载体两性电解质（一系列不同 pI 值的小分子合成分子）在电场作用下于凝胶中建立稳定的 pH 梯度。蛋白质在此梯度中迁移，直至达到其净电荷为零的 pH 值——即其等电点——便停止移动，形成一个锐利的条带。

IEF offers extremely high resolution and can distinguish between proteins that differ by as little as 0.01 pH units in pI. It is often the first dimension in two‑dimensional electrophoresis and is also used analytically in quality control of therapeutic proteins.

等电聚焦具有极高的分辨率，能够区分 pI 仅相差 0.01 pH 单位的蛋白质。它常作为双向电泳中的第一向，也在治疗性蛋白质的质量控制分析中使用。

8. Two‑Dimensional Electrophoresis (2‑DE) | 双向电泳

2‑DE combines isoelectric focusing and SDS‑PAGE to separate proteins by two independent properties: isoelectric point (first dimension) and molecular weight (second dimension). The protein sample is first run on an IEF strip; the strip is then equilibrated with SDS, placed on top of an SDS‑PAGE gel, and electrophoresed perpendicularly.

双向电泳将等电聚焦与 SDS‑PAGE 结合，按两个独立的性质——等电点（第一向）和分子量（第二向）——分离蛋白质。先用 IEF 胶条对蛋白质样品进行分离；随后用 SDS 平衡胶条，将其置于 SDS‑PAGE 凝胶顶部进行垂直电泳。

The resulting 2‑D map reveals hundreds to thousands of protein spots, each representing a unique protein species. This technique is a powerful tool in proteomics for comparing protein expression profiles between healthy and diseased cells.

得到的二维图谱显示出数百至数千个蛋白质点，每个点代表一种独特的蛋白质分子。该技术在蛋白质组学中是强大的工具，可用于比较健康细胞与病变细胞之间的蛋白质表达谱。

9. Capillary Electrophoresis | 毛细管电泳

Capillary electrophoresis (CE) performs electrophoretic separation in a narrow fused‑silica capillary (internal diameter 20–100 µm). The small dimensions allow rapid, high‑resolution separations with minute sample volumes. An important phenomenon in CE is electroosmotic flow (EOF): the negatively charged inner wall of the capillary attracts a layer of cations, which drag the entire fluid towards the cathode.

毛细管电泳在细内径石英毛细管（内径 20–100 µm）中进行电泳分离。小尺寸使得分离快速、分辨率高且样品需求量极少。毛细管电泳中一个重要现象是电渗流（EOF）：带负电的毛细管内壁吸引一层阳离子，从而拖动整个流体向阴极移动。

Detection is typically performed on‑column by UV absorbance or laser‑induced fluorescence. CE is widely applied in pharmaceutical analysis, forensic DNA profiling, and chiral separations of enantiomers. The technique encompasses several modes, including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), and capillary gel electrophoresis (CGE).

检测通常在柱上进行，采用紫外吸光度或激光诱导荧光。毛细管电泳广泛应用于药物分析、法医 DNA 分型以及对映体的手性分离。该技术包含多种模式，如毛细管区带电泳、胶束电动色谱和毛细管凝胶电泳。

10. Visualisation and Analysis of Electrophoretograms | 电泳图谱的可视化与分析

After electrophoretic separation, the separated components must be visualised. For proteins, common stains include Coomassie Brilliant Blue R‑250 (sensitivity ~100 ng) and the more sensitive silver stain. Fluorescent stains such as SYPRO Ruby offer even greater sensitivity and a broader linear dynamic range. Nucleic acids are typically stained with ethidium bromide (now largely replaced by safer alternatives like SYBR Safe) which intercalates between base pairs and fluoresces under UV light.

电泳分离后，需对分离的组分进行可视化。对于蛋白质，常用染色剂包括考马斯亮蓝 R‑250（灵敏度约 100 ng）和更灵敏的银染。SYPRO Ruby 等荧光染色剂灵敏度更高且线性动态范围更宽。核酸通常用溴化乙锭（现已大量被 SYBR Safe 等更安全的替代品取代）染色，该染料插入碱基对之间并在紫外光下发出荧光。

The gel image is recorded with a CCD camera or a scanner, and the band intensities are quantified by densitometry software. In DNA fragment length analysis, the migration distance is compared with that of a DNA ladder to estimate size. For proteins, standard marker proteins are used to construct a molecular weight calibration curve.

凝胶图像由 CCD 相机或扫描仪记录，条带强度通过光密度软件进行定量。在 DNA 片段长度分析中，将迁移距离与 DNA ladder 比较以估算大小。对于蛋白质，则用标准蛋白标记绘制分子量校正曲线。

11. Applications of Electrophoresis in Chemistry and Biology | 电泳在化学与生物学中的应用

• DNA analysis: restriction mapping, PCR product verification, DNA fingerprinting in forensic science, and genetic testing.

  DNA 分析：限制性酶切图谱、PCR 产物验证、法医学中的 DNA 指纹图谱以及基因检测。

• Protein biochemistry: purity checking, subunit composition via SDS‑PAGE, and western blotting for immunodetection of specific antigens.

  蛋白质生物化学：纯度检查、通过 SDS‑PAGE 确定亚基组成，以及用于特异性抗原免疫检测的免疫印迹（western blotting）。

• Clinical diagnostics: haemoglobin variants (e.g., sickle cell disease) are identified by electrophoresis, and serum protein electrophoresis helps diagnose multiple myeloma and liver disorders.

  临床诊断：通过电泳鉴别血红蛋白变异体（如镰状细胞病），血清蛋白电泳有助于诊断多发性骨髓瘤和肝病。

• Pharmaceutical industry: characterisation of biotherapeutics, detection of charge variants in monoclonal antibodies, and quality control of recombinant proteins.

  制药工业：生物治疗药物的表征、单克隆抗体电荷变异体的检测以及重组蛋白的质量控制。

• Food and environmental analysis: detection of genetically modified organisms (GMOs) and identification of fish or meat species via protein profiling.

  食品与环境分析：检测转基因生物，以及通过蛋白质图谱鉴别鱼或肉类物种。

12. Summary of Key Points | 关键点总结

Electrophoresis is a versatile and indispensable separation technique relying on the movement of charged molecules in an electric field. The choice of medium — agarose or polyacrylamide gel — and the use of modifiers like SDS or pH gradients allow separation by size, charge, or isoelectric point. From the simple agarose gel in a school laboratory to high‑resolution capillary electrophoresis in a pharmaceutical lab, electrophoresis remains fundamental across chemistry, biology, and medicine.

电泳是一种多功能且不可或缺的分离技术，其基础是带电分子在电场中的移动。通过选择介质——琼脂糖或聚丙烯酰胺凝胶——以及使用 SDS 或 pH 梯度等改良剂，可实现按大小、电荷或等电点的分离。从学校实验室中的简易琼脂糖凝胶，到制药实验室中的高分辨率毛细管电泳，电泳始终是化学、生物学和医学领域中的基石。

Key concepts to remember: mobility depends on charge, size, and shape; SDS‑PAGE separates proteins by molecular weight alone; IEF separates by pI; and 2‑DE combines both orthogonal dimensions. The principles of electrophoresis also underpin modern capillary techniques, enabling ultra‑sensitive and rapid analyses.

需牢记的核心概念：迁移率取决于电荷、大小和形状；SDS‑PAGE 仅按分子量分离蛋白质；等电聚焦按等电点分离；双向电泳则将两个正交的维度结合。电泳的原理也是现代毛细管技术的基础，可实现超高灵敏度和快速分析。

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