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)

ensayo de cambio en la corrida electroforética (emsa)

El análisis de cambio de movilidad electroforética (EMSA) es un procedimiento bioquímico utilizado para aclarar la unión entre proteínas y ácidos nucleicos. En este ensayo se mezclan un ácido nucleico radiomarcado y prueba de la proteína. Enlace se determina mediante electroforesis en gel que separa componentes basados en la masa, carga y conformación.
Este video muestra los conceptos de EMSA y un procedimiento general, incluyendo detección, encuadernación, electroforesis y preparación del gel y de la proteína. En este video las aplicaciones incluyen el análisis de enzimas de remodelación de la cromatina, una EMSA modificado que incorpora biontinylation y el estudio de los sitios de los reguladores de la respuesta bacteriana de Unión.
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EMSA, el análisis de cambio de movilidad electroforética, también conocido como el análisis de cambio de gel, es un procedimiento bioquímico versátil y sensible. EMSA aclara unión entre proteínas y ácidos nucleicos mediante la detección de un cambio de bandas en electroforesis en gel.
Este video describe los principios de la EMSA, proporciona un procedimiento general y habla de algunas aplicaciones.
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<div class="chapter_text" id="script_1">
Replicación del ADN, transcripción y reparación, así como procesamiento de RNA es procesos bioquímicos todos críticos. Todos implican la unión entre proteínas y ácidos nucleicos. Muchos trastornos y enfermedades graves se asocian a modificaciones en este enlace. EMSA es una técnica para determinar cualitativamente si una proteína específica se une a un ácido nucleico específico. En primer lugar, se etiqueta el ácido nucleico, generalmente con fósforo-32 radioactivo, para crear una punta de prueba. Luego la proteína de prueba y sonda de ácido nucleico se mezclan. Cuando una proteína se une a una sonda de ácido nucleico, el complejo resultante tiene mayor masa y una conformación diferente que el ácido nucleico solo.
Una vez enlazado, los complejos se analizan con electroforesis en gel. En esta técnica, un campo eléctrico fuerza macromoléculas para migrar a través de una matriz de gel. Los componentes separan en base a masa, carga y conformación. Electroforesis puede separar complejos proteína-DNA de sondas no consolidadas. Puesto que tienen conformaciones y diferentes masas, que migran por el gel a diferentes ritmos y separar. La separación se detecta fácilmente, gracias a la presencia de fósforo radiactivo y prueba con éxito, la proteína se une al ácido nucleico determinado. Para verificar la identificación de la proteína, un "supercambio" emplea un anticuerpo con una conocida afinidad a la proteína. Esto tiene la ventaja de desplazar aún más el complejo, aumentando la resolución.
Ahora que hemos visto los principios, vamos a ver en el laboratorio.
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Para comenzar el procedimiento, la proteína debe estar aislada. Para ello, se utilizan técnicas de biología molecular para expresar la proteína en las células y luego purificar.
El ácido nucleico es amplificado y etiquetado para crear una punta de prueba. Etiquetado se realiza a través de la incubación durante 10 minutos con dCTP con fósforo-32 radioactivo. Un banco de trabajo de seguridad de radiación y equipos de protección se requiere.
Luego se prepara el gel. El gel debe ser no-desnaturalizar, para evitar que la proteína alterar la conformación y potencialmente desvinculación de la sonda durante la electroforesis. Geles de poliacrilamida tienen tamaños de poro de 5 a 20 nm y son útiles para las sondas cortas hasta 100 pares de bases en longitud. Geles de agarosa tienen tamaños de poro de 70-700 nm y son útiles para las sondas más grandes.
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Con la proteína y la sonda de ácido nucleico ya preparados, procedemos a atar. Se prepara una solución de tampón TRIS y se añaden la proteína y la sonda. El pH debe ser similar a las condiciones fisiológicas y concentración de la sal suficiente para impedir que la proteína formando lazos débiles con los ácidos nucleicos no objetivo. La reacción procede por 20-30 min a 4 ° C.
El siguiente paso es la electroforesis. Se utiliza un tampón de baja fuerza iónica y pH similar a la utilizada en la reacción de enlace. Produce un "efecto jaulas" que estabiliza los complejos, aumenta la movilidad y reduce la generación de calor. Después de la electroforesis, los componentes del gel se transfieren a papel de filtro. En un cuarto oscuro, el papel de filtro luego se expone a la película. Si la proteína se une a la sonda, dos regiones diferentes de etiquetado será visibles en la transferencia. Que representa el complejo y aparte una que representa la punta de prueba independiente. La separación demuestra que la proteína unida con éxito para el ácido nucleico.
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Ahora que hemos visto lo básico, vamos a ver algunas aplicaciones.
La cromatina es el complejo firmemente lleno de ADN y proteínas que se encuentran las células eucariotas. Remodelación de cromatina enzimas modifican la estructura para abrir la DNA transcripción. Como esto cambia la movilidad del complejo, EMSA puede utilizarse para explorar la actividad de la enzima.
Un enfoque alternativo para etiquetado toma ventaja de la interacción entre la DNA y la enzima metiltransferasa. Un cofactor puede ser modificado para enlazar permanentemente a la DNA vía metiltransferasa. En lugar de etiquetar con fósforo-32, el cofactor es conjugado con biotina, que es ventajoso ya que no es radiactivo. Porque el cofactor es específico en su fijación, es relevante para la genotipificación y detección de metilación genes. Los biotinilated los ácidos nucleicos son detectados a través de fluorescencia ULTRAVIOLETA.
Cuando estímulos ambientales activan cinasas de la histidina, se fosforila un "regulador de respuesta". Esto a su vez se une al DNA, que afectan a la transcripción, que puede ser estudiada por EMSA. Por ejemplo, un regulador de respuesta de Desulfovibrio vulgaris fue demostrado para enlazar con el gen de interés. EMSA se utilizó para verificar que el enlace tuvo lugar.
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<div class="chapter_text" id="script_5">
Sólo ha visto video en lo análisis de cambio de movilidad electroforética de Zeus. Ahora debe comprender sus principios de operación, los pasos de su procedimiento y sus principales parámetros de funcionamiento.
¡Gracias por ver!
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Ensayo de cambio en la corrida electroforética (EMSA)

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a ...

EMSA, el ensayo de cambio de movilidad electroforética, también conocido como ensayo de cambio de gel, es un procedimiento bioquímico versátil y sensible. EMSA dilucida la unión entre proteínas y ácidos nucleicos mediante la detección de un cambio en las bandas en la electroforesis en gel. Este video describe los principios de EMSA, proporciona un procedimiento general y analiza algunas aplicaciones. La replicación, transcripción y reparación del ADN, así como el procesamiento del ARN, son procesos bioquímicos críticos. Todos ellos implican la unión entre proteínas y ácidos nucleicos. Muchas enfermedades y trastornos graves se asocian con modificaciones en esta unión. EMSA es una técnica para determinar cualitativamente si una proteína específica se une a un ácido nucleico específico. Primero, el ácido nucleico se marca, generalmente con fósforo radiactivo 32, para crear una sonda. A continuación, se mezclan la proteína de prueba y la sonda de ácido nucleico. Cuando una proteína se une a una sonda de ácido nucleico, el complejo resultante tiene mayor masa y una conformación diferente a la del ácido nucleico solo. Una vez unidos, los complejos se analizan con electroforesis en gel. En esta técnica, un campo eléctrico obliga a las macromoléculas a migrar a través de una matriz de gel. Los componentes se separan en función de la masa, la carga y la conformación. La electroforesis puede separar los complejos proteína-ADN de las sondas no unidas. Dado que tienen diferentes masas y conformaciones, migrarán a través del gel a diferentes velocidades y se separarán. La separación es fácilmente detectable, gracias a la presencia del fósforo radiactivo, y demuestra que la proteína se une con éxito al ácido nucleico dado. Para verificar la identificación de la proteína, un "ensayo de superdesplazamiento" utiliza un anticuerpo con una afinidad conocida por la proteína. Esto tiene el beneficio adicional de cambiar aún más el complejo, aumentando la resolución. Ahora que hemos visto los principios, vamos a verlo en el laboratorio. Para comenzar el procedimiento, se debe aislar la proteína. Para ello, se utilizan técnicas de biología molecular para expresar la proteína en las células, y luego purificarla. El ácido nucleico se amplifica y se marca para crear una sonda. El marcaje se realiza mediante incubación durante 10 min con dCTP que contiene fósforo radiactivo-32. Se requiere un banco de trabajo a prueba de radiación y equipo de protección. A continuación, se prepara el gel. El gel debe ser no desnaturalizante, para evitar que la proteína altere la conformación y potencialmente se desvincule de la sonda durante la electroforesis. Los geles de poliacrilamida tienen tamaños de poro de 5 a 20 nm y son útiles para sondas cortas de hasta 100 pares de bases de longitud. Los geles de agarosa tienen tamaños de poro de 70-700 nm y son útiles para sondas más grandes. Con la sonda de proteínas y ácidos nucleicos ya preparada, procedemos a la unión. Se prepara una solución tampón TRIS y se añaden la proteína y la sonda. El pH debe ser similar a las condiciones fisiológicas y la concentración de sal suficiente para evitar que la proteína forme enlaces débiles con ácidos nucleicos no objetivo. La reacción continúa durante 20-30 min a 4 ?C.El siguiente paso es la electroforesis. Se utiliza un tampón de baja fuerza iónica y un pH similar al utilizado en la reacción de unión. Produce un "efecto de jaula" que estabiliza los complejos, aumenta la movilidad y reduce la generación de calor. Después de la electroforesis, los componentes del gel se transfieren al papel de filtro. En una habitación oscura, el papel de filtro se expone a la película. Si la proteína se une a la sonda, dos regiones marcadas distintas serán visibles en la transferencia. Uno que representa el complejo y otro separado que representa la sonda no enlazada. La separación demuestra que la proteína se unió con éxito al ácido nucleico. Ahora que hemos visto el procedimiento básico, veamos algunas aplicaciones. La cromatina es el complejo compacto de ADN y proteínas que se encuentran en las células eucariotas. Las enzimas remodeladoras de la cromatina modifican la estructura para abrir el ADN a la transcripción. A medida que esto cambia la movilidad del complejo, EMSA se puede utilizar para explorar la actividad de unión de la enzima. Un enfoque alternativo para el marcaje aprovecha la interacción entre el ADN y la enzima metiltransferasa. Un cofactor puede ser modificado para unirse permanentemente al ADN a través de la metiltransferasa. En lugar de marcarse con fósforo-32, el cofactor se conjuga con biotina, lo cual es ventajoso porque no es radiactivo. Debido a que el cofactor es específico del sitio en su unión, es relevante para el genotipado, la detección de metilación y la entrega de genes. Los ácidos nucleicos biotinilados se detectan mediante fluorescencia ultravioleta. Cuando los estímulos ambientales activan las histidina quinasas, un ?regulador de la respuesta? está fosforilado. Esto, a su vez, se une al ADN, afectando la transcripción, que puede ser estudiada por EMSA. Por ejemplo, ¿un regulador de respuesta en? Se demostró que Desulfovibrio vulgaris se une al gen de interés. Se utilizó la EMSA para verificar que la unión se había llevado a cabo. Acabas de ver el vídeo de JoVE sobre el ensayo de cambio de movilidad electroforética. Ahora debe comprender sus principios de funcionamiento, los pasos de su procedimiento y sus principales parámetros de funcionamiento. ¡Gracias por mirar!

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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

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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...

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co-immunoprecipitation and pull-down assays

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f&#246;rster resonance energy transfer (fret)

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biochemistry

Esta colección presenta varios métodos de purificación comúnmente usados, como la cromatografía de afinidad y métodos analíticos, como MALDI-TOF. Además, los videos muestran métodos para evaluar la interacción y la función de diversas biomoléculas como en la coprecipitación y el etiquetado metabólico.

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

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Matter and Energy in Organisms and Ecosystems

HS-LS1-6.

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HS-LS3-1.

Earth and Space Sciences

Human Sustainability

HS-ESS3-3.

Overview

Principles of EMSA

Protein and Gel Preparation

Binding, Electrophoresis, and Detection

Applications

Summary

Characteristics of Life

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Cell Structure

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Nucleic Acids

DNA as a Genetic Template

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An Overview of Epigenetics

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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 ...

detecting-environmental-microorganisms-with-polymerase-chain-reaction

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
Polymerase chain reaction (PCR) is ...

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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