Electrophoresis: Principles, Methods and Applications | 电泳：原理、方法与应用

📚 Electrophoresis: Principles, Methods and Applications | 电泳：原理、方法与应用

Electrophoresis is a separation technique widely used in chemistry, biochemistry and molecular biology to separate charged particles such as ions, amino acids, proteins and nucleic acids under the influence of an electric field. In the A-level curriculum, electrophoresis is often linked to the analysis of amino acids, proteins and DNA fragments, helping students understand how charge, size and buffer pH affect the movement of molecules.

电泳是一种广泛应用于化学、生物化学和分子生物学的分离技术，用于在电场作用下分离离子、氨基酸、蛋白质和核酸等带电粒子。在 A-level 课程中，电泳常与氨基酸、蛋白质和 DNA 片段的分析联系起来，帮助学生理解电荷、分子大小和缓冲液 pH 如何影响分子的运动。

1. What Is Electrophoresis? | 什么是电泳？

Electrophoresis is the migration of charged particles through a stationary medium, usually a gel or a buffer solution, when a potential difference is applied across two electrodes. Positively charged cations move towards the cathode, which is the negative electrode, while negatively charged anions move towards the anode, which is the positive electrode.

电泳是指当在两个电极之间施加电势差时，带电粒子在静止介质（通常是凝胶或缓冲溶液）中发生迁移的过程。带正电的阳离子向阴极（负极）移动，而带负电的阴离子向阳极（正极）移动。

The technique is particularly useful for separating macromolecules that cannot easily be separated by simple filtration or distillation because they are heat-sensitive or present in small quantities. Electrophoresis allows separation based on differences in charge-to-size ratio rather than boiling point or solubility.

该技术特别适用于分离那些难以通过简单过滤或蒸馏分离的大分子，因为这些大分子往往对热敏感或含量很少。电泳是基于电荷与大小之比的差异进行分离，而不是依据沸点或溶解度的差异。

2. Basic Principle of Separation | 基本分离原理

When a charged particle is placed in an electric field of strength E, it experiences an electrical force equal to the product of its net charge q and the field strength. This force is opposed by frictional drag from the surrounding medium. The particle accelerates briefly until the two forces balance, after which it moves at a constant velocity.

当带电粒子置于电场强度为 E 的电场中时，它会受到一个等于其净电荷 q 与场强乘积的电力。该力受到周围介质摩擦阻力的阻碍。粒子短暂加速，直到两个力达到平衡，之后以恒定速度运动。

The migration rate of a spherical particle can be described approximately by the following relationship:

球形粒子的迁移速率可近似用以下关系描述：

μ = q / (6πηr)

Here μ is the electrophoretic mobility, q is the net charge on the particle, η is the viscosity of the medium and r is the effective radius of the particle. This relationship shows that highly charged and small particles move faster, whereas large particles experience more frictional resistance and move more slowly.

其中 μ 为电泳迁移率，q 为粒子的净电荷，η 为介质黏度，r 为粒子的有效半径。该关系表明，电荷多且体积小的粒子移动较快，而较大的粒子受到更大的摩擦阻力，移动较慢。

3. Key Factors Affecting Electrophoresis | 影响电泳的关键因素

Several experimental variables determine how well molecules separate by electrophoresis. Understanding these factors is important for interpreting gel images and explaining why bands appear at particular positions.

若干实验变量决定了电泳分离分子的效果。理解这些因素对于解释凝胶图像以及说明条带为何出现在特定位置非常重要。

Net charge: The greater the magnitude of charge, the larger the electrical force and the faster the migration under the same field.
Particle size and shape: Compact, small molecules move more quickly through the gel pores. Larger or more irregular molecules are slowed by sieving effects.
Electric field strength: A higher voltage increases migration speed, but excessive voltage can generate heat and cause band distortion.
Buffer pH: pH controls the ionisation state of acidic and basic groups, so it changes the net charge of molecules such as amino acids and proteins.
Support medium: The pore size and composition of the gel affect the sieving of macromolecules.
Temperature: High temperature lowers buffer viscosity and increases mobility, but can denature proteins or melt agarose gels.

净电荷：电荷越大，相同电场下电力越大，迁移越快。
粒子大小与形状：紧密的小分子在凝胶孔隙中移动更快；较大或形状不规则的分子因筛分效应而变慢。
电场强度：电压越高，迁移速度越快，但电压过高会产生热量并导致条带变形。
缓冲液 pH：pH 控制酸性和碱性基团的电离状态，从而改变氨基酸和蛋白质等分子的净电荷。
支持介质：凝胶孔径和组成影响大分子的筛分效果。
温度：高温降低缓冲液黏度并提高迁移率，但可能使蛋白质变性或使琼脂糖凝胶熔化。

4. Support Media: Agarose and Polyacrylamide Gels | 支持介质：琼脂糖凝胶与聚丙烯酰胺凝胶

Electrophoresis is usually carried out in a gel rather than in free solution because the gel minimises diffusion and provides a sieving matrix. The two most common gel materials are agarose and polyacrylamide.

电泳通常在凝胶中而非自由溶液中进行，因为凝胶可以最大限度地减少扩散并提供筛分基质。两种最常见的凝胶材料是琼脂糖和聚丙烯酰胺。

Agarose gels are prepared from agarose polysaccharide extracted from seaweed. They have relatively large pores and are mainly used for separating large nucleic acid fragments such as DNA or RNA molecules. By changing the agarose concentration, the pore size can be adjusted; lower concentrations allow larger fragments to pass more easily.

琼脂糖凝胶由从海藻中提取的琼脂糖多糖制备。其孔径相对较大，主要用于分离 DNA 或 RNA 等大核酸片段。通过改变琼脂糖浓度可以调节孔径；浓度越低，较大片段越容易通过。

Polyacrylamide gels are formed by polymerising acrylamide and a cross-linker. They have smaller, more controllable pores and give higher resolution for small DNA fragments, RNA and especially proteins. Polyacrylamide gel electrophoresis is commonly abbreviated as PAGE.

聚丙烯酰胺凝胶由丙烯酰胺与交联剂聚合而成。其孔径更小、更可控，对小的 DNA 片段、RNA，尤其是蛋白质具有更高的分辨率。聚丙烯酰胺凝胶电泳通常缩写为 PAGE。

Feature	Agarose gel	Polyacrylamide gel
Typical use	DNA and RNA fragments	Proteins and small nucleic acids
Pore size	Larger, adjustable	Smaller, highly controllable
Resolution	Lower	Higher

5. Electrophoresis of Amino Acids | 氨基酸的电泳

Amino acids are ideal examples for studying electrophoresis because their net charge depends strongly on the pH of the surrounding buffer. In acidic conditions, the amino group is protonated to -NH₃⁺ and the carboxyl group may be largely unionised, giving the amino acid a net positive charge. In basic conditions, the carboxyl group is deprotonated to -COO⁻ and the amino group is mostly neutral, giving a net negative charge.

氨基酸是研究电泳的理想例子，因为它们的净电荷很大程度上取决于周围缓冲液的 pH。在酸性条件下，氨基质子化为 -NH₃⁺，羧基大部分未电离，使氨基酸带净正电荷。在碱性条件下，羧基去质子化为 -COO⁻，氨基大多呈中性，使氨基酸带净负电荷。

At a specific pH known as the isoelectric point, pI, an amino acid exists as a zwitterion with equal numbers of positive and negative charges. Its net charge is zero, so it does not migrate in an electric field. Different amino acids have different pI values, and therefore they move at different rates and in different directions at a given buffer pH.

在称为等电点 pI 的特定 pH 下，氨基酸以兼性离子形式存在，正负电荷数目相等。其净电荷为零，因此在电场中不发生迁移。不同氨基酸具有不同的 pI 值，因此在给定缓冲液 pH 下，它们以不同速率和不同方向移动。

For example, at pH 6, glutamate, with its acidic side chain, is negatively charged and moves towards the anode, whereas arginine, with its basic side chain, is positively charged and moves towards the cathode.

例如，在 pH 6 时，带有酸性侧链的谷氨酸带负电并向阳极移动，而带有碱性侧链的精氨酸带正电并向阴极移动。

6. Protein Electrophoresis and Native PAGE | 蛋白质电泳与 Native PAGE

Proteins are large macromolecules with many ionisable side chains, so their net charge is the sum of contributions from all acidic and basic residues. In native PAGE, proteins are separated in their folded, biologically active state without denaturing agents. Their migration depends on charge, size and shape simultaneously.

蛋白质是大型大分子，具有许多可电离侧链，因此其净电荷是所有酸性残基和碱性残基贡献的总和。在 Native PAGE 中，蛋白质在不含变性剂的折叠、具有生物活性状态下分离。它们的迁移同时取决于电荷、大小和形状。

Because native proteins retain their three-dimensional structure, two proteins of the same molecular mass may migrate differently if their shapes or charge distributions differ. This can be useful for studying protein complexes or enzyme activity, but it makes molecular mass estimation difficult.

由于天然蛋白质保留其三维结构，两个分子质量相同的蛋白质如果形状或电荷分布不同，可能会以不同方式迁移。这对研究蛋白质复合物或酶活性很有用，但会使分子质量估算变得困难。

7. SDS-PAGE: Size-Based Protein Separation | SDS-PAGE：基于大小的蛋白质分离

SDS-PAGE is the most common method for separating proteins according to their molecular mass. The protein sample is first treated with sodium dodecyl sulfate, SDS, a negatively charged detergent. SDS binds to the polypeptide chain, denatures the protein and coats it with a uniform negative charge proportional to its length.

SDS-PAGE 是根据分子质量分离蛋白质的最常用方法。蛋白质样品首先用十二烷基硫酸钠（SDS）处理，SDS 是一种带负电的去污剂。SDS 与多肽链结合，使蛋白质变性，并使其带有与链长成正比的均匀负电荷。

A reducing agent such as dithiothreitol or β-mercaptoethanol is often added to break disulfide bonds, allowing the protein to unfold completely. Under these conditions, the intrinsic charge of the protein becomes negligible compared with the large negative charge from SDS, so all proteins have a similar charge-to-mass ratio.

通常会加入二硫苏糖醇或 β-巯基乙醇等还原剂来断裂二硫键，使蛋白质完全展开。在这些条件下，蛋白质固有的电荷与 SDS 带来的大量负电荷相比可以忽略不计，因此所有蛋白质具有相似的荷质比。

Separation then occurs mainly through the sieving effect of the polyacrylamide gel. Smaller proteins migrate faster because they pass through the pores more easily, while larger proteins are retarded. By comparing the distance travelled by unknown proteins with the distances travelled by molecular mass markers, the molecular mass of each protein can be estimated.

随后，分离主要通过聚丙烯酰胺凝胶的筛分效应发生。较小的蛋白质因更容易通过孔隙而迁移更快，而较大的蛋白质则受到阻滞。通过将未知蛋白质的迁移距离与分子质量标记物进行比较，可以估算每种蛋白质的分子质量。

After separation, proteins are usually stained with Coomassie Brilliant Blue or silver stain so that the bands become visible. The band intensity can give a rough indication of protein abundance.

分离后，蛋白质通常用考马斯亮蓝或银染法染色，使条带可见。条带强度可以粗略反映蛋白质的丰度。

8. Isoelectric Focusing | 等电聚焦

Isoelectric focusing, IEF, separates proteins according to their isoelectric points. A stable pH gradient is established in a polyacrylamide gel using special carrier ampholytes. When a protein is placed in this gradient and an electric field is applied, it migrates through regions of different pH.

等电聚焦（IEF）根据蛋白质的等电点进行分离。使用特殊的两性电解质在聚丙烯酰胺凝胶中建立稳定的 pH 梯度。当蛋白质置于该梯度中并施加电场时，它会在不同 pH 区域中迁移。

As a protein moves towards the electrode, it encounters a pH that changes its ionisation state. When it reaches the region where the local pH equals its pI, its net charge becomes zero, so it stops moving. Each protein therefore becomes focused into a sharp band at its own isoelectric point.

当蛋白质向电极移动时，会遇到改变其电离状态的 pH。当它到达局部 pH 等于其 pI 的区域时，其净电荷变为零，因此停止移动。因此，每种蛋白质都会在其自身的等电点处聚焦成清晰的条带。

Isoelectric focusing is often combined with SDS-PAGE in two-dimensional gel electrophoresis, abbreviated 2D-PAGE, which separates proteins first by charge and then by mass. This gives very high resolution and can separate thousands of proteins in a single gel.

等电聚焦常与 SDS-PAGE 结合用于二维凝胶电泳，简称 2D-PAGE，先按电荷分离蛋白质，再按质量分离。这种方法分辨率很高，可以在单块凝胶中分离数千种蛋白质。

9. DNA Electrophoresis and Applications | DNA 电泳及其应用

DNA molecules are negatively charged at neutral pH because of the phosphate groups in their sugar-phosphate backbone. When a DNA sample is loaded into an agarose gel and a voltage is applied, all fragments migrate towards the positive anode. Since DNA fragments have a uniform charge-to-mass ratio, separation is based almost entirely on size.

DNA 分子在中性 pH 下因糖-磷酸骨架中的磷酸基团而带负电。当 DNA 样品加入琼脂糖凝胶并施加电压时，所有片段都向正极阳极迁移。由于 DNA 片段具有均匀的荷质比，分离几乎完全基于大小进行。

Smaller DNA fragments migrate faster through the agarose pores, while larger fragments move more slowly. A DNA ladder containing fragments of known sizes is run alongside the sample, allowing the size of unknown fragments to be determined by comparing band positions.

较小的 DNA 片段通过琼脂糖孔隙迁移较快，而较大的片段移动较慢。含有已知大小片段的 DNA 梯度标记物与样品同时电泳，通过比较条带位置可以确定未知片段的大小。

DNA electrophoresis is essential in DNA fingerprinting for forensic analysis and paternity testing, in checking the success of polymerase chain reaction, PCR, and in restriction enzyme mapping. It is also used to separate DNA fragments for sequencing and cloning experiments.

DNA 电泳在法医分析和亲子鉴定的 DNA 指纹分析、检查聚合酶链反应（PCR）是否成功以及限制性内切酶图谱分析中必不可少。它还用于分离 DNA 片段以进行测序和克隆实验。

After electrophoresis, DNA is stained with ethidium bromide or safer dyes such as SYBR Safe, which intercalate between base pairs and fluoresce under ultraviolet light. The resulting bands can be photographed and analysed.

电泳后，DNA 用溴化乙锭或 SYBR Safe 等更安全的染料染色，这些染料插入碱基对之间并在紫外光下发出荧光。得到的条带可以拍照并进行分析。

10. Visualisation, Recording and Interpretation | 显色、记录与结果解读

Because most biological molecules are colourless, the separated bands must be visualised after electrophoresis. The choice of staining method depends on the type of molecule being analysed.

由于大多数生物分子是无色的，电泳后必须使分离的条带可视化。染色方法的选择取决于所分析分子的类型。

Proteins: Coomassie Brilliant Blue, silver stain or fluorescent dyes.
DNA/RNA: Ethidium bromide, SYBR Safe or other nucleic acid stains.
Amino acids: Ninhydrin spray can be used for detection after paper or thin-layer electrophoresis.

蛋白质：考马斯亮蓝、银染或荧光染料。
DNA/RNA：溴化乙锭、SYBR Safe 或其他核酸染料。
氨基酸：纸电泳或薄层电泳后可用茚三酮喷雾检测。

Interpreting a gel requires comparing the positions of sample bands with those of standard markers. The relative mobility, Rf, can be calculated by dividing the distance moved by the band by the distance moved by the dye front or by a reference marker. This helps normalise results between different gels.

解读凝胶需要将样品条带的位置与标准标记物进行比较。相对迁移率 Rf 可通过条带迁移距离除以染料前沿或参照标记物迁移距离来计算。这有助于在不同凝胶之间对结果进行归一化。

A common mistake is to assume that the most intense band always represents the largest molecule. In fact, band position indicates size or charge, while intensity indicates the amount of material, so interpretation must be based on both position and intensity.

一个常见错误是认为最强的条带总是代表最大的分子。事实上，条带位置表示大小或电荷，而强度表示物质的量，因此必须结合位置和强度进行解读。

11. Experimental Method Summary | 实验方法总结

A typical electrophoresis experiment involves several key steps. First, the gel is prepared by dissolving agarose or polymerising acrylamide in a buffer and pouring it into a casting tray with a comb to create sample wells. Once the gel has set, it is placed in an electrophoresis tank and covered with running buffer.

典型的电泳实验包括几个关键步骤。首先，制备凝胶：将琼脂糖溶解或使丙烯酰胺在缓冲液中聚合，倒入带有梳子的制胶盘中以形成样品孔。凝胶凝固后，将其放入电泳槽中并加入电泳缓冲液覆盖。

Samples are mixed with a loading buffer that contains glycerol to help them sink into the wells and a tracking dye such as bromophenol blue or xylene cyanol to monitor the progress of the run. An electric potential is then applied across the gel, and the charged molecules migrate through the matrix.

样品与含有甘油的加样缓冲液混合，甘油有助于样品沉入孔中，还含有溴酚蓝或二甲苯蓝等示踪染料以监测电泳进程。然后在凝胶两端施加电压，带电分子穿过基质迁移。

After a suitable time, the power is switched off, the gel is removed and stained, and the bands are recorded with a camera or scanner. Safety precautions are important, especially when using electrical equipment, ultraviolet light and nucleic acid stains.

适当时间后，关闭电源，取出凝胶并染色，用相机或扫描仪记录条带。安全预防措施非常重要，尤其是在使用电气设备、紫外灯和核酸染料时。

12. Exam Tips and Common Misconceptions | 考试技巧与常见误区

In A-level examinations, questions on electrophoresis often ask students to predict the direction of migration of an amino acid at a given pH, explain why SDS is added to protein samples, or describe how DNA fragments of different sizes are separated. Clear use of scientific terminology is essential.

在 A-level 考试中，关于电泳的题目常要求学生预测氨基酸在给定 pH 下的迁移方向，解释为什么在蛋白质样品中加入 SDS，或描述不同大小的 DNA 片段如何被分离。清晰使用科学术语至关重要。

Misconception: ‘All molecules move towards the positive electrode.’ Correct: Only negatively charged particles move towards the anode; positively charged particles move towards the cathode.
Misconception: ‘SDS gives proteins a positive charge.’ Correct: SDS is an anionic detergent that gives proteins a uniform negative charge.
Misconception: ‘Larger molecules always move faster.’ Correct: In a sieving gel, larger molecules usually move more slowly, provided charge-to-mass ratios are similar.
Misconception: ‘At its pI, a protein is uncharged.’ More precise: It has a net charge of zero but still contains equal numbers of positive and negative local charges.

误区：‘所有分子都向正极移动。’ 正确：只有带负电的粒子向阳极移动；带正电的粒子向阴极移动。
误区：‘SDS 使蛋白质带正电。’ 正确：SDS 是一种阴离子去污剂，使蛋白质带均匀的负电荷。
误区：‘较大的分子总是移动更快。’ 正确：在筛分凝胶中，如果荷质比相似，较大的分子通常移动较慢。
误区：‘蛋白质在其 pI 时没有电荷。’ 更准确地说：其净电荷为零，但仍含有等量的正电荷和负电荷局部基团。

When answering exam questions, always refer to charge, size and pH, and link observations to the underlying chemistry of ionisable groups. This will help you gain maximum marks for explanation-style questions.

回答考试问题时，始终提及电荷、大小和 pH，并将观察结果与可电离基团的基础化学联系起来。这将有助于你在解释类题目中获得满分。

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Electrophoresis: Principles, Methods and Applications | 电泳：原理、方法与应用