十二烷基硫酸钠聚丙烯酰胺凝胶电泳，也称SDS-PAGE，是一种被广泛使用，仅根据分子量大小来分离蛋白质混合物的技术。 阴离子去污剂SDS，在变性的线性蛋白表面沿长度均匀分布使其带电。 将它们上样到聚丙烯酰胺凝胶后， 施加电压，这些表面覆盖SDS的蛋白将被分开。电场作为驱动力，牵引SDS结合的蛋白朝阳极移动，分子量大的蛋白将比小的蛋白移动慢。为了判断蛋白的大小，已知分子量大小的蛋白质标准也会和样品一起上样并在同等条件下跑胶。
本短片介绍SDS-PAGE技术, 首先将解释其背后的原理，然后演示每一步的操作过程。视频还将讨论实验中的各种参数，如聚丙烯酰胺浓度，和用于跑胶的电压。还会介绍电泳之后的考马斯亮蓝和银染色方法，以及其他电泳技术，如双向凝胶电泳。
SDS-PAGE (十二烷基硫酸钠聚丙烯酰胺凝胶)
SDS-PAGE是一种被许多研究人员用来根据分子量大小分离蛋白质混合物的技术。成功完成该技术是如免疫杂交 等多种蛋白质分析方法的重要前提。而该技术本身也是一个用来测定蛋白质大小和纯度的有用工具。
SDS-PAGE系统的主要成分
要了解SDS-PAGE技术，首先要知道它的主要组成成分。SDS-PAGE是十二烷基硫酸钠聚丙烯酰胺凝胶电泳的缩写。其中的第一部分SDS, 也就是十二烷基硫酸钠, 是一种阴离子去污剂，它由带负电的亲水基团和不带电的疏水长链组成。疏水长链覆盖在蛋白表面，与其分子大小成比例，每克蛋白 可结合1.4克SDS。它是电场中蛋白质依其分子大小分离的驱动力。
聚丙烯酰胺凝胶电泳使用由聚丙烯酰胺制作的水凝胶。聚丙烯酰胺是聚合物，它形成一种非常均一的基质，蛋白质可在其中迁移。凝胶的浓度越高，施加电场后蛋白质在其中移动的速度越慢。这种使用均一电场来使得物质移动的过程称之为电泳。
SDS-PAGE样品准备
SDS-PAGE可用来分离许多不同来源的蛋白，包括培养的细胞，组织，血液，尿液和酵母。这些不同来源的蛋白首先要通过匀浆，裂解，离心来使其与其他细胞组分分开。蛋白分离后，为确保样品上样量相同，要用二喹啉甲酸BCA，光度法检测，比较样本与血球蛋白标准样中的蛋白量来测定蛋白浓度。
添加上样缓冲液也是很重要的一个步骤。上样缓冲液有三个主要作用，第一，由于其中的SDS和其他变性剂，它将变性蛋白，也就是将蛋白结构打开变成完全线性的氨基酸链。第二，上样缓冲液中含有甘油，可确保样本上样到胶孔中后不会浮起来。最后，多数商品化的上样缓冲液都含有染料，如溴酚蓝，可帮助在电泳过程中检测跑胶的进度。
加入上样缓冲液后，混合样品，煮沸5分钟。这样可保证蛋白质中坚固的二硫键被变性剂β-巯基乙醇还原打开。双硫键打开后，SDS可更均一地覆盖在蛋白的表面。
接下来，迅速离心样品然后就可上样到凝胶中进行电泳。
上样和跑胶
上样前，先要将凝胶电泳系统装好。首先是购买或者配制聚丙烯酰胺凝胶。由于丙烯酰胺是神经毒素，对大脑会有损伤，购买预制凝胶现在越来越普遍。凝胶夹板中有用来上样的胶孔。
把凝胶固定好后，在内槽和外槽中盛满与制备凝胶同样离子浓度的缓冲液。这样会形成一个电流环路，流畅地从负极，通过凝胶，再到正极。
接下来上样样品，之后通常会上样分子量标准样。跑胶时，标准样会分散开形成一组可见的已知分子量大小的蛋白带。这些蛋白带最后可用于计算每个蛋白的大小。
上样完成后，电泳盒的正极和负极连接到电源来持续不断提供稳定的电压。通常在样品进入胶之前，使用60伏电压。然后将电压增加到200伏，根据凝胶的大小和浓度不同以及目的蛋白的大小，跑胶时间可为30分钟到1小时。
应用
电泳完成后，取出凝胶夹板并将其打开取出凝胶。再用典型的蛋白染料，如考马斯亮蓝或银染色来染色胶，使得凝胶中的蛋白带显色。考马斯亮蓝最低可检测到50纳克的蛋白量，而银染的灵敏度可达1纳克蛋白量。
双向凝胶电泳中，样品在凝胶中以两种不同属性分开，每次一个方向。首先，样品上样到固相的pH梯度胶中，根据等电点不同将 蛋白分开。再将pH胶中的蛋白变性，放置在聚丙烯酰胺凝胶上方并用新鲜的胶溶液固定好，然后用SDS-PAGE进行第二向的分离。
双向凝胶电泳并不都是通过等电点来分离，但它是最普遍的方式。双向凝胶电泳是一种宝贵的工具，可帮助了解蛋白复合物和亚细胞结构。
跑胶完成后，通常的做法是将胶上的蛋白转移到由PVDF或尼龙制成的膜上进行分析。你可以用特定的抗体杂交膜来寻找目的蛋白。这里显示的是一个典型的免疫杂交结果，科研人员使用了3种不同的信号放大技术来判断哪种能最好地检测到梯度稀释溶液中的Pit-1蛋白。
结语
您刚观看的是JoVE关于使用SDS-PAGE分离蛋白质的视频。现在您应该对如何使用该强大工具分析蛋白质分子大小的步骤有所了解。我们一如既往地感谢您的观看。
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