双向[凝胶]电泳（英语：Two-dimensional [gel] electrophoresis）是一种等电聚焦电泳与SDS-PAGE相结合，分辨率更高的蛋白质电泳检测技术。双向电泳后的凝胶经染色蛋白呈现二维分布图，水平方向反映出蛋白在等电点上的差异，而垂直方向反映出它们在分子量上的差别。所以双向电泳可以将分子量相同而等电点不同的蛋白质以及等电点相同而分子量不同的蛋白质分开。双向电泳是快速成长的蛋白质组学技术中最流行最通用的蛋白质分离方法。目前2D-PAGE能够在同一块凝胶上同步检测和定量数千个蛋白质。

Two-dimensional gel electrophoresis, (2-DE or 2D electrophoresis), is a form of gel electrophoresis commonly used to isolate the proteins in a sample for further characterization by mass spectroscopy. Mixtures of proteins are separated in the 1st dimension by their charge (isoelectric focusing (IEF)). Then in the 2nd dimension, proteins are separated by their size (molecular weight). This video shows the part 1- protein extraction and precipitation.

样品处理

一般性原则

样品制备是双向电泳中最为关键的一步，这一步处理的好坏将直接影响 2-DE 结果。目前并没有一个通用的制备方法，尽管处理方法是多种多样，但都遵循几个基本的原则：

1. 尽可能的提高样品蛋白的溶解度，抽提最大量的总蛋白，减少蛋白质的损失；
2. 减少对蛋白质的人为修饰；
3. 破坏蛋白质与其他生物大分子的相互作用，并使蛋白质处于完全变性状态。

根据这一原则，样品制备需要以下几种主要的试剂：

1. 离液剂(chaotropes),主要包括尿素（Urea）和硫脲（thiourea） ；
2. 表面活性剂（sufactants），也称去垢剂，早期常使用 NP-40、TritonX-100 等非离子去垢剂，近几年较多的改用如 CHAPS与 Zwittergent 系列等双性离子去垢剂；
3. 还原剂（reducing agents） ，最常用的是二硫苏糖醇（DTT） ，也有用二硫赤藓糖醇（DTE）以及磷酸三丁酯（TBP）等。
4. 蛋白酶抑制剂（如 EDTA、PMSF or Protease inhibitorcocktails）
5. 核酸酶。
6. 当然，也可以选择性的加入Tris-base。

样品的来源不同，其裂解的缓冲液也各不相同。通过不同试剂的合理组合，以达到对样品蛋白的最大抽提。在对样品蛋白质提取的过程中，必须考虑到去除影响蛋白质可溶性和 2DE 重复性的物质，比如核酸、脂、多糖等大分子以及盐类小分子。大分子的存在会阻塞凝胶孔径，盐浓度过高会降低等电聚焦的电压，甚至会损坏 IPG 胶条；这样都会造成 2-DE 的失败。样品制备的失败很难通过后续工作的完善或改进获得补偿。

核酸的去除可采用超声或核酸酶处理，超声处理应控制好条件，并防止产生泡沫；而加入的外源核酸酶则会出现在最终的 2D 胶上。脂类和多糖都可以通过超速离心除去。透析可以降低盐浓度，但时间太长；也可以采取凝胶过滤或沉淀/重悬法脱盐，但会造成蛋白质的部分损失。 因此，处理方法必须根据不同的样品、所处的状态以及实验目的和要求来进行选择。

样品制备程序

培养细胞样品处理方法

培养动物组织细胞由于没有细胞壁，因此可以将细胞收集下来，直接加入裂解缓冲液（Lysis buffer）抽提总蛋白。裂解缓冲液有多种配方，主要有如下成份：

• (A) 7M Urea，2M Thiourea，4％（w/v）CHAPS，40mM Tris-Base，40mM DTT，2％ Pharmalyte pH 3-10.

其他常用的裂解缓冲液如下：

• (B) 9.5M urea, 2%(w/v) CHAPS, 0.8%(w/v) Phamarlyte pH3-10, 1%(w/v) DTT and 5mM Pefabloc proteinase inhibitor;
• (C) 加入 0.3-1% SDS在95°C 煮样品 5mins，冷却后加入至少5倍体积的（A）或（B）裂解液。

总蛋白抽提程序：

1. 培养细胞的收集；
2. 用磷酸缓冲液（PBS）洗细胞 3 次（室温，1000g，各 2min）；
3. 将细胞分装到 1.5ml Eppendof 管中，吸干残留的 PBS；
4. 加入裂解缓冲液（1.5×10⁶ 个细胞大约加入 100µL裂解液） ，在室温振荡 1h，使其充分溶解；
5. 4°C，40,000g，离心 1h；
6. 吸取上清并用Brandford法定量蛋白，然后分装至Eppendof管里保存在-78°C备用。

组织样品处理方法

对大多数从动物或植物组织里提取总蛋白质而言，同样没有一种通用的程序。但遵循的原则基本相同。下面列出一种对植物树叶总蛋白的方法。
三氯醋酸－丙酮沉淀法提取植物树叶总蛋白程序：

1. 在液氮中研碎叶片；
2. 悬浮于含 10％三氯醋酸（TCA）和 0.07%β-巯基乙醇(可用DTT替代)的丙酮溶液在－20 °C的冰浴；
3. 让蛋白质沉淀过夜然后离心（4°C，40,000g, 1h） ，弃上清；
4. 重悬沉淀浮于含 0.07%β-巯基乙醇的冰预冷丙酮溶液里；
5. 离心（4°C，40,000g, 1h）后真空干燥沉淀；
6. 用（A）或（B）裂解液溶解沉淀，离心（4°C，40,000g, 1h）。
7. Brandford法定量蛋白，然后分装至Eppendof管里保存在-78°C备用。

超速离心法：

1. 取材；
2. 用研钵在液氮冷冻条件下将样品研成粉末，每1g样品加入 0.5ml裂解液，使用组织匀浆器匀浆 30 s；
3. 组织悬液 15°C，10 000×g离心 10 min；
4. 上清液 4°C，150 000×g超速离心 45 min；
5. 小心避开上层漂浮的脂质层，吸取离心上清6°C 40,000g再次离心 50 min；
6. 取离心上清。Bradford法定量，分装后置–75°C保存。

文献报道较多的裂解液配方

注：CA、蛋白酶抑制剂混合物和DTT在临用前加入。其中 C-F是分步法提取的4种裂解液配方, C适于提取水溶性蛋白，D是经典配方，E适于提取膜蛋白配方，F适于提取难溶的沉淀蛋白。
Lysis buffer A
9 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.5% CA and a cocktail of protease inhibitors

新鲜配制，或-20°C冻存。使用时加入cocktail蛋白酶抑制剂和DTT储存液至DTT终浓度为1%（例如，200 μl裂解液中加入13 μl 15.5×的DTT储存液）。
Lysis buffer B
7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% w/v DTT, 0.5% CA and a cocktail of protease inhibitors.
Lysis buffer C
40 mM Tris-base (pH 9.5) in ultrapure H₂O
Lysis buffer D
8 M urea, 4% CHAPS, 40 mM Tris(base), 40 ml

新鲜配制，或-20°C冻存。使用时加入cocktail蛋白酶抑制剂和DTT储存液至DTT终浓度为1%（例如，200 μl裂解液中加入13 μl 15.5×的DTT储存液）。
Lysis buffer E
5 M urea, 2 M thiourea, 2% SB 3-10, 2% CHAPS, 1% w/v DTT, 0.5% CA and a cocktail of protease inhibitors

新鲜配制，或-20°C冻存。使用时加入cocktail蛋白酶抑制剂和DTT储存液至DTT终浓度为1%（例如，200 μl裂解液中加入13 μl 15.5×的DTT储存液）。
Lysis buffer F
100 μL SDS sample solution (1% w/v SDS, 0.375 M Tris-HCl, pH 8.8, 50 mM DTT, 25% v/v glycerol)

Protein extraction is the starting step for almost all protein purification. Depending on the cell type selected as the source for target protein expression, cellular extracts contain largeamounts of nucleic acid, ribosomal material, lipids, dispersedcell wall polysaccharide, carbohydrates, small molecules, and
thousands of unwanted proteins (1). Because purification of protein is one of the most important subjects in protein research to understand their function, as they may partly or completely be involved in any DNA synthesis activity (2).Isolation and recovery of a single protein from this complex mixture of macro-molecules presents considerable challenges(1). Protein can be extracted by a few methods such as detergent
lysis, shearing force, treatment with low ionic salt (salting out), and rapid changes in pressure, which aimed to weaken and break the membranes surrounding the cell to allow proteins to escape (3). The big and possibly most important of these challenges is generation of a cellular extract that can be efficiently manipulated in downstream processes without inactivation or degradation of labile protein targets (1).
Some factors should be considered when handling proteins. Normally, protein extraction is performed at a very low temperature (4 degree C) as proteins are easily denatured once they are released from the cells. Buffer condition is one of the major factors that need to be considered. Specific buffer conditions are recommended to be maintained because of thesensitivity of proteins toward environmental pH changes (4). The purity of water will affect the yield of end products as unpurified water contains a lot of microorganisms or proteases that will result in protein degradation (4). Protein inhibitor, which may exist in solution or buffers, causes the hydrolyzation of proteins. Detergent, another significant factor that cannot be neglected in purification of protein, consists of a hydrophobic portion of a linear or branched hydrocarbon “tail” and a hydrophilic “head” (4). Reducing agents will be added into solution or
buffer for protein extraction and purification to avoid the lost of activity of proteins or enzymes which is caused by oxidization.

Visual Protein’s ExtractPRO Protein Extraction Reagent is a new formula reagent for extracting most of bio-samples and reduce the most contaminations including DNA, RNA and cell debris. The ExtractPRO Protein Extraction Reagent not only effectively extract proteins from cultured cells and mammalia tissues without using protease inhibitor, but also easily precipitate proteins after protein extraction (Figure 1, 2). ExtractPRO Protein Extraction Reagent omit the tedious and time-consuming traditional methods, and can maintain protein profile after long-term sample preservation in ExtractPRO reagent.

A. Sample preparation
A1. Cultured cells
1. Harvest cells (106~107) by centrifugation, scraping or trypsin treatment.
2. Discard the supernatant carefully.
3. Wash cells in 5 mL of ice-cold PBS and discard the supernatant after centrifugation for 5 minutes at 250 × g. Repeat this step three times.
4. Add 1 mL of ExtractPRO Reagent.
5. Transfer the cells to a 1.5 mL microcentrifuge tube.

Note: Disruption of some yeast and bacterial cells may require the use of a power homogenizer.

A2. Tissues
1. Add 1 mL of ExtractPRO Reagent in 50~100 mg of tissue sample.
2. Homogenize tissue sample using a Tissue Grinder or power homogenizer.
3. Transfer the tissue sample to a 1.5 mL microcentrifuge tube.

Note: The sample volume should not exceed 10% of the volume of ExtractPRO Reagent used for homogenization.

B. Protein extraction
1. Vortex vigorously for 15 minutes.
2. Centrifuge the tube at 12,000 × g for 10 minutes at 4°C.
3. Transfer the supernatant to a new tube
4. Add 200 μL of chloroform. Shake for 15 seconds.
5. Incubate for 3 minutes at room temperature.
6. Centrifuge at 12,000 × g for 15 minutes at 4°C.
Note: ExtractPRO Reagent should be separated three phase (aqueous phase, interphase, organic phase from top to bottom respectively)
7. Discard aqueous phase carefully.
8. Add 300 μL of 100% ethanol. Invert for 15 seconds.
9. Centrifuge at 2,000 × g for 5 minutes at 4°C.
10. Divide equally the supernatant into two new tubes.

C. Protein precipitation
1. Add 750 μL of Isopropyl alcohol per tube. Shake for 15 seconds.
2. Incubate for 10 minutes at room temperature.
3. Centrifuge at 16,000 × g for 10 minutes at 4°C.
4. Discard supernatant carefully.
5. Add 1 mL of ice-cold acetone. Vortex the protein pellets for oneminute.
6. Centrifuge at 16,000 × g for 10 minutes at 4°C.
7. Discard acetone carefully.
8. Repeat Step 5~7 twice.
9. Evaporate residual acetone by open tube cover for few minute.Do not allow the pellet to dry out.
10. Pour the protein pellets to a 1.5 mL microcentrifuge tube usingtip.
11. Resuspend protein pellets using suitable buffer depending ondownstream experiment.