电泳迁移分析法 (EMSA)
English
Condividere
Panoramica
电泳迁移分析法 (EMSA) 是用来阐明蛋白质与核酸之间的绑定的生化过程。在这种测定方法中混合了放射性标记的核酸和蛋白质测试。绑定通过凝胶电泳分离组件基于质量、 电荷和构象。
该视频演示 EMSA 和一般的程序，包括凝胶和蛋白质的制备、 绑定、 电泳，检测的概念。覆盖在此视频的应用程序包括的染色质重塑的酶，包含 biontinylation，改性 EMSA 的分析与研究结合部位的细菌响应监管机构。
EMSA，电泳迁移分析法，也被称为凝胶转移检测，是一个多才多艺和敏感的生化过程。EMSA 通过检测转移乐队在凝胶电泳中阐明了蛋白质和核酸之间的绑定。
这个视频描述了 EMSA 的原则，所提供的一般过程，并探讨了一些应用程序。
DNA 复制、 转录、 和维修，以及 RNA 加工是所有关键的生化过程。他们都涉及到蛋白质与核酸之间的绑定。许多严重的疾病和障碍都与此绑定中修改。EMSA 是定性地确定是否某一特定蛋白绑定到特定的核酸技术。首先，核酸标记，通常与放射性磷-32，以创建探测器。然后测试蛋白质和核酸探针混合。当蛋白质与核酸探针结合时，由此产生的复杂有更大的质量和不同的构象比独自核酸。
绑定后，用凝胶电泳分析了配合物。在这种技术，电场力大分子通过凝胶基质迁移。组件单独的基于的质量、 电荷和构象。电泳法能从绑定探针分离蛋白-DNA 复合物。因为它们具有不同的群众和构象，他们将通过以不同的速率凝胶迁移和独立。分离很容易发现，由于放射性磷的存在，并证明了蛋白质成功绑定到给定的核酸。若要验证蛋白的鉴定，”supershift”使用免疫抗体与蛋白已知亲和力。这有进一步转移的复杂性，提高分辨率的额外的好处。
现在，我们已经看到的原则，让我们看看在实验室里。
开始执行程序，这种蛋白质必须隔离。为此，分子生物学技术用于表达蛋白在细胞中，然后净化。
核酸是放大和贴上标签，创建探测器。标签是通过培养 10 分钟完成与筒含放射性磷-32。辐射安全工作台和防护设备的要求。
然后制备凝胶。这种凝胶需要非变性，以防止改变构象和潜在解除绑定从探针在电泳过程中的蛋白质。聚丙烯酰胺凝胶中长度有孔隙大小的 5 到 20 nm 和非常有用的短探针到 100 个碱基对。琼脂糖凝胶有 70 700 毫微米的孔径大小，可用于较大的探头。
蛋白质和核酸探针现在做好了准备，与我们进行绑定。三缓冲溶液编写和添加蛋白质和探针。Ph 值应该是类似于生理条件和盐浓度足以防止形成弱键与非靶核酸蛋白。使反应进行 20-30 分钟在 4 ° c。
下一步是电泳。利用低离子强度和 ph 值类似，在结合反应中使用的缓冲区。它产生一种”锁定效应”，稳定配合物，增加流动性，减少产生的热量。后电泳，凝胶组分转移到滤纸上。在一个黑暗的房间里，过滤纸然后暴露电影。如果蛋白绑定到探头，两个不同的标记的区域将在转让中可见。一个表示复杂和一个单独的表示未绑定的探针。这种蛋白质成功绑定到核酸分离演示
现在，我们已经看到的基本程序，让我们看看一些应用程序。
染色质是紧凑的复杂的 DNA 和蛋白质发现真核细胞。染色质重塑酶修改要打开到转录 DNA 的结构。随着这变化的复杂流动性，可以用 EMSA 来探索结合活性的酶。
标签的另一种方法充分利用了甲基转移酶与 DNA 相互作用。可以修改辅助因子，以永久绑定到 DNA 甲基转移酶通过。而不是标签与磷-32，辅酶共轭与生物素结合，这是有利的因为它不是放射性。因为辅酶是特定于站点在其附件中，它是有关的基因分型、 甲基化检测和基因传递。Biotinilated 核酸通过紫外荧光进行检测。
当环境刺激激活组氨酸激酶时，磷酸化”反应调节”。这反过来将绑定到 DNA，影响转录，可通过 EMSA 研究。例如，表现出弧菌寻常型反应调节要绑定到感兴趣的基因。EMSA 用于验证绑定发生。
你刚看了朱庇特的视频上电泳迁移分析法。现在，您应该了解其原理的操作，其过程和其主要的操作参数中的步骤。
谢谢观赏 ！
Procedura
Divulgazioni
Trascrizione
EMSA, the electrophoretic mobility shift assay, also known as the gel shift assay, is a versatile and sensitive biochemical procedure. EMSA elucidates binding between proteins and nucleic acids by detecting a shift in bands in gel electrophoresis.
This video describes the principles of EMSA, provides a general procedure, and discusses some applications.
DNA replication, transcription, and repair, as well as RNA processing are all critical biochemical processes. They all involve binding between proteins and nucleic acids. Many serious diseases and disorders are associated with modifications in this binding. EMSA is a technique for qualitatively determining whether a specific protein binds to a specific nucleic acid. First, the nucleic acid is labeled, usually with radioactive phosphorus-32, to create a probe. Then the test protein and nucleic acid probe are mixed. When a protein binds to a nucleic acid probe, the resulting complex has greater mass and a different conformation than the nucleic acid alone.
Once bound, the complexes are analyzed with gel electrophoresis. In this technique, an electric field forces macromolecules to migrate through a gel matrix. The components separate based on mass, charge, and conformation. Electrophoresis can separate protein-DNA complexes from unbound probes. Since they have different masses and conformations, they will migrate through the gel at different rates and separate. The separation is easily detected, thanks to the presence of the radioactive phosphorus, and proves the protein successfully binds to the given nucleic acid. To verify the identification of the protein, a “supershift assay” uses an antibody with a known affinity to the protein. This has the added benefit of further shifting the complex, increasing resolution.
Now that we’ve seen the principles, let’s see it in the lab.
To begin the procedure, the protein must be isolated. To do this, molecular biology techniques are used to express the protein in cells, and then purify.
The nucleic acid is amplified and labeled to create a probe. Labeling is done through incubation for 10 min with dCTP containing radioactive phosphorus-32. A radiation-safe workbench and protective equipment are required.
The gel is then prepared. The gel needs to be non-denaturing, to prevent the protein from altering conformation and potentially unbinding from the probe during electrophoresis. Polyacrylamide gels have pore sizes of 5 to 20 nm and are useful for short probes up to 100 base pairs in length. Agarose gels have pore sizes of 70-700 nm and are useful for larger probes.
With the protein and nucleic acid probe now prepared, we proceed to binding. A TRIS buffer solution is prepared and the protein and probe are added. The pH should be similar to physiological conditions, and salt concentration sufficient to prevent the protein from forming weak bonds with non-target nucleic acids. The reaction proceeds for 20-30 min at 4 °C.
The next step is electrophoresis. A buffer of low ionic strength and a pH similar to that used in the binding reaction is utilized. It yields a “caging effect” that stabilizes complexes, increases mobility, and reduces heat generation. After electrophoresis, the components of the gel are transferred onto filter paper. In a dark room, the filter paper is then exposed to film. If the protein binds to the probe, two distinct labeled regions will be visible in the transfer. One representing the complex, and a separate one representing the unbound probe. The separation demonstrates that the protein successfully bound to the nucleic acid.
Now that we’ve seen the basic procedure, let’s look at some applications.
Chromatin is the tightly packed complex of DNA and proteins found eukaryotic cells. Chromatin-remodeling enzymes modify the structure to open the DNA to transcription. As this changes the mobility of the complex, EMSA can be used to explore the binding activity of the enzyme.
An alternate approach to labeling takes advantage of the interaction between DNA and the methyltransferase enzyme. A cofactor can be modified to bind permanently to DNA via methyltransferase. Rather than labeling with phosphorus-32, the cofactor is conjugated to biotin, which is advantageous because it’s not radioactive. Because the cofactor is site-specific in its attachment, it’s relevant to genotyping, methylation detection, and gene delivery. The biotinilated nucleic acids are detected through ultraviolet fluorescence.
When environmental stimuli activate histidine kinases, a “response regulator” is phosphorylated. This in turn binds to DNA, affecting transcription, which can be studied by EMSA. For instance, a response regulator in Desulfovibrio vulgaris was demonstrated to bind to the gene of interest. EMSA was used to verify that the binding took place.
You’ve just watched JoVE’s video on the electrophoretic mobility shift assay. You should now understand its principles of operation, the steps in its procedure, and its major operating parameters.
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