Name: detecting protein-dna binding by emsa
Uploaded: 2023-04-30T06:31:38.000Z
Duration: 6 min 57 s
Channel: JoVE

K12 Standards

new-york-state-science-learning-standards

New York State Science Learning Standards

High School

Life Science

Structure and Function

HS-LS1-1.

electrophoretic-mobility-shift-assay-emsa

Electrophoretic Mobility Shift Assay (EMSA)

电泳迁移分析法 (emsa)

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。
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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。
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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。
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<div class="chapter_text" id="script_2">
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。
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<div class="chapter_text" id="script_3">
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
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<div class="chapter_text" id="script_4">
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。
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<div class="chapter_text" id="script_5">
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 ！
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电泳迁移分析法 (EMSA)

电泳迁移率变动分析（EMSA）是一种用于阐明蛋白质与核酸之间结合关系的生物化学实验方法。在该实验中，一个……

EMSA（电泳迁移率偏移分析），也称为凝胶迁移分析，是一种多功能且灵敏的生物化学实验方法。EMSA通过检测凝胶电泳中条带的迁移偏移，揭示蛋白质与核酸之间的结合情况。本视频介绍了凝胶迁移阻滞实验（EMSA）的原理，提供了一般性操作流程，并讨论了部分应用。DNA 复制、转录和修复，以及 RNA 加工，均为关键的生化过程。这些过程均涉及蛋白质与核酸之间的结合。许多严重的疾病和疾患均与这种结合的改变有关。EMSA 是一种用于定性判断特定蛋白质是否与特定核酸结合的技术。首先，用放射性磷-32 通常对核酸进行标记，以制备探针。然后将待测蛋白质与核酸探针混合。当蛋白质与核酸探针结合时，所形成的复合物比单独的核酸具有更大的质量及不同的构象。结合后，利用凝胶电泳对复合物进行分析。该技术通过电场驱动大分子穿过凝胶基质，根据其质量、电荷和构象实现分离。电泳可将蛋白质- DNA 复合物与未结合的探针分离开来。由于它们具有不同的质量和构象，因此在凝胶中的迁移速率不同，从而实现分离。由于探针中含有放射性磷，分离结果易于检测，从而证明该蛋白质能够成功结合目标核酸。为了进一步验证蛋白质的身份，可采用“超迁移实验”（supershift assay），即加入对目标蛋白质具有已知亲和力的抗体。这种方法还能进一步改变复合物的迁移位置，提高分辨率。现在我们已经了解了原理，接下来让我们在实验室中进行演示。开始该实验步骤前，必须先分离目标蛋白。为此，需采用分子生物学技术在细胞中表达该蛋白，随后进行纯化。核酸经过扩增并标记以制备探针。标记过程通过与含有放射性磷-32的dCTP孵育10分钟完成。操作需要在防辐射工作台中进行，并佩戴适当的防护装备。然后制备凝胶。凝胶应为非变性条件，以防止蛋白质在电泳过程中发生构象改变并可能从探针上解离。聚丙烯酰胺凝胶的孔径大小为5至20 nm，适用于长度不超过100个碱基对的短探针。琼脂糖凝胶的孔径范围为70–700 nm，适用于较大的探针。蛋白质和核酸探针制备完成后，我们接下来进行结合反应。配制TRIS缓冲液溶液，并将蛋白质和探针加入其中。pH值应接近生理条件，盐浓度需足够高，以防止蛋白质与非靶标核酸形成弱结合。反应在4 ?C下进行20-30分钟。下一步是电泳。使用低离子强度且pH值与结合反应中相同的缓冲液。这种方法会产生“笼蔽效应”，可稳定复合物、提高迁移率，并减少热量产生。电泳完成后，将凝胶中的组分转移至滤纸上。在暗室中，将滤纸进行胶片曝光。如果蛋白质与探针发生结合，在转移产物中将可见两个不同的标记区域：一个代表复合物，另一个代表未结合的探针。该分离结果表明蛋白质已成功结合核酸。了解了基本步骤后，让我们来看一些应用实例。染色质是真核细胞中由DNA和蛋白质紧密包裹形成的复合物。染色质重塑酶可修饰其结构，使DNA解聚以利于转录。由于这一过程会改变复合物的迁移能力，因此可利用EMSA来研究该酶的结合活性。另一种标记方法利用了DNA与甲基转移酶之间的相互作用。可通过甲基转移酶将经过修饰的辅因子永久结合到DNA上。该方法不使用磷-32进行标记，而是将辅因子与生物素偶联，从而避免了放射性，具有明显优势。由于辅因子的结合具有位点特异性，因此该技术适用于基因分型、甲基化检测以及基因递送。生物素标记的核酸可通过紫外荧光进行检测。当环境刺激激活组氨酸激酶时，一个“反应调节蛋白”会被磷酸化。该调节蛋白继而结合DNA，影响转录过程，这一过程可通过电泳迁移率变动分析（EMSA）进行研究。例如，已证明在脱硫弧菌（Desulfovibrio vulgaris）中，一个反应调节蛋白能够结合目标基因，该结合事件通过EMSA得以验证。您刚刚观看了 JoVE 关于电泳迁移率变动分析的视频。现在，您应该理解该实验的基本原理、操作步骤以及主要的运行参数。感谢观看！

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detecting protein-dna binding by emsa

Electrophoretic mobility shift assay, or EMSA, is a biochemical method used to detect binding between proteins and nucleic acids. It is also called a gel shift assay. The test compares how a labeled nucleic acid moves through a gel before and after it binds a protein.

EMSA is used to see whether a specific protein binds to a specific nucleic acid. First, the nucleic acid is labeled to make a probe. The transcript describes radioactive phosphorus-32 as a common label. The probe is then mixed with the test protein, and any binding changes the size and shape of the complex.

The bound and unbound molecules are then separated by gel electrophoresis. An electric field pulls macromolecules through a gel matrix. They separate by mass, charge, and conformation. Protein-DNA complexes move differently from free probe, so the shifted band shows that binding occurred. A supershift assay can also confirm the protein’s identity. In that step, an antibody with known affinity binds the protein and shifts the complex even further.

The lab procedure begins by isolating the protein. Molecular biology methods are used to express the protein in cells and then purify it. The nucleic acid is amplified and labeled to form the probe. Because phosphorus-32 is radioactive, a radiation-safe workbench and protective equipment are required during labeling.

The gel must be non-denaturing so the protein does not change conformation or fall off the probe during electrophoresis. Polyacrylamide gels have pore sizes of about 5 to 20 nm and are used for short probes up to 100 base pairs long. Agarose gels have larger pores, about 70 to 700 nm, and are used for larger probes. After the protein and probe are ready, they are mixed in TRIS buffer for binding.

The binding reaction uses a pH close to physiological conditions. The salt concentration must be high enough to reduce weak, non-target binding. The reaction runs for 20 to 30 min at 4 °C. Electrophoresis is then done in a buffer with low ionic strength and a similar pH. This creates a caging effect that helps stabilize complexes, increases mobility, and reduces heat generation.

After the gel runs, the components are transferred onto filter paper. The paper is exposed to film in a dark room. If binding occurred, two labeled regions appear: one for the protein-nucleic acid complex and one for the unbound probe. That separation shows the protein successfully bound the nucleic acid.

EMSA is useful in several research areas. Chromatin-remodeling enzymes can be studied because they change chromatin structure and affect DNA mobility. A modified EMSA can use biotinylated nucleic acids instead of radioactive labels. This approach is site-specific and useful for genotyping, methylation detection, and gene delivery. It is also detected through ultraviolet fluorescence.

The assay can also help study bacterial response regulators. When environmental stimuli activate histidine kinases, a response regulator becomes phosphorylated and binds DNA to affect transcription. The transcript gives an example from Desulfovibrio vulgaris, where EMSA was used to confirm binding to a gene of interest. EMSA therefore provides a sensitive way to study protein-nucleic acid interactions and their role in DNA replication, transcription, repair, and RNA processing.

Detecting Protein-DNA Binding by EMSA

dialysis: diffusion based separation

Dialysis for Buffer Exchange in Biochemistry

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two-dimensional gel electrophoresis

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electrophoretic mobility shift assay (emsa)

Electrophoretic mobility shift assay, or EMSA, is a biochemical method used to detect binding between proteins and nucleic acids. It is also called a gel shift assay. The test compares how a labeled nucleic acid moves through a gel before and after it binds a protein. EMSA is used to see whether a specific protein binds to a specific nucleic acid. First, the nucleic acid is labeled to make a probe. The transcript describes radioactive phosphorus-32 as a common label. The probe is then mixed...

photometric protein determination

Measuring Protein Concentration with Absorbance

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density gradient ultracentrifugation

Sucrose Gradient Separation in an Ultracentrifuge

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co-immunoprecipitation and pull-down assays

co-immunoprecipitation-and-pull-down-assays

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reconstitution of membrane proteins

Membrane Proteins in Liposome Models

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biochemistry

本系列介绍了常用的纯化方法, 如亲和层析, 以及分析方法, 如 MALDI。此外, 视频展示了评估生物相互作用和功能的方法, 如 co-immunoprecipitation 和代谢标记。

Learn how EMSA detects protein-DNA binding with labeled probes, gel electrophoresis, and band shifts. See the steps and common applications.

Detecting Protein-DNA Binding by EMSA | JoVE K12

matter-and-energy-in-organisms-and-ecosystems

Matter and Energy in Organisms and Ecosystems

HS-LS1-6.

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HS-LS3-1.

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Human Sustainability

HS-ESS3-3.

Overview

Principles of EMSA

Protein and Gel Preparation

Binding, Electrophoresis, and Detection

Applications

Summary

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The Nucleus

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The Central Dogma

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Nucleic Acids

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Structure of a Gene

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Translation

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Protein and Protein Structures

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An organism&#8217;s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its ...

An Introduction to Cell Death

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An Overview of Genetic Engineering

Genetic engineering &#8211; the process of purposefully altering an organism&#8217;s DNA &#8211; has been used to create powerful research tools and model ...

An Introduction to Cell Division

Cell division is the process by which a parent cell divides and gives rise to two or more daughter cells. It is a means of reproduction for single-cell ...

An Overview of Genetics and Disease

Many human diseases are associated with mutations or variations in genetic sequences. Some of these genetic variants are heritable, passed down from ...

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An Introduction to Cellular and Molecular Neuroscience

Cellular and molecular neuroscience is one of the newest and fastest growing subdisciplines in neuroscience. By investigating the influences of genes, ...

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...

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An Introduction to Developmental Genetics

Development is the complex process through which a single-celled embryo transforms into a multicellular organism. Developmental processes are guided by ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

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An Introduction to Molecular Developmental Biology

Molecular signals play a major role in the complex processes occurring during embryonic development. These signals regulate activities such as cell ...

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Bacterial Transformation: The Heat Shock Method

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of ...

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An Introduction to Saccharomyces cerevisiae

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Cell Cycle Analysis

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An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

Chromatin Immunoprecipitation

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex ...

An Overview of Epigenetics

Since the early days of genetics research, scientists have noted certain heritable phenotypic differences that are not due to differences in the ...

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Community DNA Extraction from Bacterial Colonies

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Traditional methods of analysis ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, ...

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Embryonic Stem Cell Culture and Differentiation

Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-renewal ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

Enzyme Assays and Kinetics

Enzyme kinetics describes the catalytic effects of enzymes, which are biomolecules that facilitate chemical reactions necessary for living organisms. ...

DNA Ligation Reactions

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond.  An enzyme known as a ligase ...

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Explant Culture for Developmental Studies

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo ...

DNA Methylation Analysis

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the ...

Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...

Density Gradient Ultracentrifugation

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact ...

F&#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent ...

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Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz
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Gel Purification

Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...

Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells.

Detecting Protein-DNA Binding by EMSA

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