Protein Separation & Purification Techniques | 蛋白质分离纯化常用技术

📚 Protein Separation & Purification Techniques | 蛋白质分离纯化常用技术

In cell biology and biotechnology, a single protein rarely exists in its pure state: a typical cell contains thousands of different proteins. Purifying a target protein from this complex mixture — while preserving its structure and function — is a fundamental experimental challenge. It is also an essential skill for A-level practical assessments, where you need to explain not only how each technique works, but also why a particular sequence of methods is chosen.

在细胞生物学和生物技术中，单一蛋白质几乎不会以纯净状态存在：一个典型的细胞含有数千种不同的蛋白质。从复杂的混合物中纯化目标蛋白、同时保持其结构与功能，是一项基础的实验挑战。这也是 A-level 实践考核的重要技能——你不仅要解释每种技术的工作原理，还要说明为什么选择特定的方法组合。

1. Core Principles of Protein Purification | 蛋白质纯化的核心原理

All purification techniques exploit one or more physical or chemical differences between the target protein and the contaminants. The most commonly used properties are solubility, molecular size, net electrical charge, and specific binding affinity. A good purification plan combines several methods, each giving a different selectivity, in a logical order.

所有纯化技术都利用目标蛋白质与杂质之间的一项或多项物理与化学差异。最常用的性质包括溶解度、分子大小、净电荷和特异性结合亲和力。一个合理的纯化方案，是把多种具有不同选择性的方法按照逻辑顺序组合起来。

The goal is to maximise purification fold (the increase in specific activity after each step) while minimising protein loss. Purity and yield are inversely related: every additional step removes contaminants but also loses some target protein. Understanding this trade-off is central to designing any purification protocol.

纯化的目标是在每一步后最大化“纯化倍数”（每一步比活性的提高），同时尽量减少蛋白质损失。纯度与得率往往互斥：每增加一步虽能去除更多杂质，但也会损失部分目标蛋白。理解这种取舍是设计任何纯化方案的核心。

2. Step One: Getting the Proteins out of Cells | 第一步：使蛋白质从细胞中释放

Purification always begins with cell lysis (also called homogenisation). The cell membranes must be broken to release the soluble proteins into an extraction buffer. The buffer is kept cold (usually 0–4 °C) to slow down proteases, and contains a suitable pH buffer plus protease inhibitors. Keeping the protein cold and buffered preserves its native conformation and biological activity.

纯化总是从细胞裂解（又称匀浆化）开始。必须破碎细胞膜，使可溶性蛋白质释放到提取缓冲液中。缓冲液需保持低温（通常 0–4 °C）以减缓蛋白酶的作用，并含有合适的 pH 缓冲剂和蛋白酶抑制剂。保持低温与缓冲环境，可以维持蛋白质的天然构象和生物活性。

Common lysis methods include: mechanical grinding with sand, high-speed blending in a homogeniser, sonication using ultrasonic waves, freeze–thaw cycling, and detergent lysis which dissolves the lipid membrane. After lysis, the suspension is centrifuged to remove cell debris and organelles, leaving a clear crude extract (supernatant) ready for further purification.

常用裂解方法包括：加砂研磨、匀浆器高速搅拌、利用超声波进行超声破碎、冻融循环，以及用去垢剂溶解脂质膜。裂解后，悬浮液经离心去除细胞碎片和细胞器，得到澄清的粗提液（上清液），供后续纯化使用。

3. Centrifugation — First Separation Step | 离心——第一步分离

Centrifugation separates particles by size and density. In differential centrifugation, the sample is spun at progressively higher speeds. Low-speed spins (about 1000 × g) sediment whole cells and nuclei; medium-speed spins sediment mitochondria; high-speed ultracentrifugation can sediment ribosomes and large protein complexes. Each pellet can be collected for further analysis.

离心按颗粒的大小和密度进行分离。在差速离心中，样品以逐步升高的转速离心。低速离心（约 1000 × g）沉淀整细胞和细胞核；中速离心沉淀线粒体；高速超速离心可沉淀核糖体和大型蛋白质复合物。每次形成的沉淀都可分别收集用于进一步分析。

In density-gradient centrifugation, the sample is layered on top of a gradient medium such as sucrose. During spinning, each particle migrates until it reaches the position where its buoyant density matches the surrounding gradient, forming sharp bands. For routine protein purification, simple centrifugation is usually only a preliminary clarifying step before chromatography.

在密度梯度离心中，样品被小心铺在梯度介质（如蔗糖）的顶部。离心过程中，各粒子迁移至与其自身浮力密度相等的梯度位置，形成清晰的条带。在常规蛋白质纯化中，简单离心通常只是层析之前的一个预澄清步骤。

4. Salting Out — Precipitation by High Salt | 盐析——高盐沉淀

Protein solubility in water depends on a shell of ordered water molecules surrounding each protein’s charged and polar groups. When a high concentration of a salt such as ammonium sulfate, (NH₄)₂SO₄, is added, the salt ions compete for water molecules and strip away this hydration shell. Exposed hydrophobic regions then cause the proteins to aggregate and precipitate — this is salting out.

蛋白质在水中的溶解度依赖于其带电基团和极性基团周围形成的有序水化层。当加入高浓度盐（如硫酸铵 (NH₄)₂SO₄）时，盐离子与水分子竞争，将水化层剥去。暴露的疏水区使蛋白质聚集而沉淀——这就是盐析。

Because different proteins precipitate at different salt concentrations, fractional precipitation is possible: you add the salt stepwise, collecting the precipitate formed at each concentration. Salting out is cheap, gentle, and is often used early in the purification process. The precipitated protein is recovered by centrifugation and redissolved in a small volume of buffer.

由于不同蛋白质在不同盐浓度下才开始沉淀，因此可实现分级沉淀：逐步加入盐，在每个浓度下收集所产生的沉淀。盐析成本低、条件温和，常用于纯化的前期阶段。沉淀的蛋白质经离心回收，再用少量缓冲液重新溶解。

5. Dialysis — Cleaning up the Sample | 透析——样品的“清洗”

Dialysis removes small molecules and exchanges buffers. The protein solution is sealed inside a semi-permeable membrane (a dialysis bag) and placed in a large volume of buffer. Small solutes such as salt ions and sugars diffuse through the pores and equilibrate with the outside buffer, while proteins, being much larger, are retained inside the bag.

透析用于去除小分子和更换缓冲液。将蛋白质溶液密封在半透膜（透析袋）内，并浸入大量缓冲液中。盐离子、糖等小溶质通过膜孔扩散并与外部缓冲液达到平衡，而分子较大的蛋白质则被截留在袋内。

The molecular weight cut-off (MWCO) of the membrane determines what can pass through; for proteins, a MWCO of 10–14 kDa is common. Dialysis is the standard way to remove ammonium sulfate

Published by TutorHao | Biology Revision Series | aleveler.com

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