Name: chip methods for dna-protein binding
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 36 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Chromatin Immunoprecipitation- ChIP

La inmunoprecipitación de cromatina, abreviada como ChIP, es una técnica para estudiar las interacciones proteína-ADN que regulan la expresión génica.
En los eucariotas, el ADN se envuelve alrededor de las proteínas histonas y forma un complejo conocido como nucleosoma, que se agrupa aún más en una estructura conocida como cromatina para ayudar en el empaquetamiento apretado del ADN en las células.
La expresión génica se regula a través de modificaciones de histonas que desenredan o tensan estas estructuras, así como proteínas que se asocian con regiones cis-reguladoras en el ADN.
En ChIP, la cromatina se descompone y se utilizan anticuerpos que se unen a las modificaciones de histonas o proteínas reguladoras para aislar las moléculas objetivo con el ADN asociado.
El primer paso en este proceso es reticular la proteína al ADN con la ayuda de un agente de reticulación, como el formaldehído, que inmoviliza la proteína en el ADN, marcando el sitio de unión de la proteína. Después de la reticulación, la cromatina se corta mecánicamente en fragmentos cortos que van de 100 a 200 pares de bases. Este proceso se conoce como X-ChIP.
Un método alternativo es el N-ChIP, en el que las nucleasas digieren directamente el ADN de la cromatina en fragmentos cortos, sin ningún tipo de reticulación previa.
La inmunoprecipitación se lleva a cabo mediante la introducción de anticuerpos que se dirigen a las proteínas reguladoras en la solución que contiene ADN cortado o digerido. Estos anticuerpos están ligados a agentes que ayudan en el aislamiento selectivo.
Un método común es vincular los anticuerpos a perlas magnéticas. Se utiliza un imán para aislar los anticuerpos junto con las moléculas unidas.
El complejo se enjuaga para eliminar cualquier contaminante vagamente asociado y las moléculas objetivo se separan con la ayuda de un detergente, como SDS.
En el caso de X-ChiP, la reticulación se invierte con la ayuda de temperaturas elevadas, y los reguladores asociados o histonas se degradan con la ayuda de proteasas, dejando atrás el ADN. 
La secuenciación del ADN asociado puede ayudar a identificar la secuencia cis-reguladora a la que se unió la proteína de interés o el gen cuya expresión está modulada por una modificación particular de histonas.

<ol>
	<li>Gade, P., & Kalvakolanu, D. V. (2012). Ensayo de inmunoprecipitación de cromatina como herramienta para analizar la actividad del factor de transcripción. Métodos en biología molecular (Clifton, N.J.), 809, 85–104. https://doi.org/10.1007/978-1-61779-376-9_6</li>
	<li>Nelson, J. D., Denisenko, O., & Bomsztyk, K. (2006). Protocolo para el método de inmunoprecipitación rápida de cromatina (ChIP). Protocolos de la Naturaleza, 1(1), 179–185. doi:10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004). Ensayo de inmunoprecipitación de cromatina. Biotécnica, 37(6), 961-969.</li>
</ol>

inmunoprecipitación de cromatina: chip

La inmunoprecipitación de cromatina, o ChIP, es una técnica basada en anticuerpos que se utiliza para identificar sitios en el ADN que se unen a factores de transcripción de interés o proteínas histonas. También ayuda a determinar el tipo de modificaciones de las histonas, como la acetilación, la fosforilación o la metilación.

Tipos de chips

ChIP se puede dividir en dos tipos: X-ChIP y N-ChIP. X-ChIP implica el entrecruzamiento in vivo de histonas y proteínas reguladoras con el ADN, la fragmentación del ADN mediante sonicación y el aislamiento de los complejos proteína-ADN inmunoprecipitándolos con anticuerpos específicos. Por otro lado, N-ChIP no implica entrecruzamiento del ADN con proteínas y la digestión se lleva a cabo mediante nucleasas.

Análisis posterior de las interacciones entre ADN y proteínas.

Después de aislar el ADN de interés, se pueden utilizar para el análisis técnicas como PCR, microarrays o Southern blot. Alternativamente, este ADN se puede utilizar para una secuenciación profunda, conocida como ChIP-Seq. ChIP se puede modificar utilizando otras metodologías para diferentes análisis, como ChIA-PET, una técnica que combina los principios de ChIP con la captura de la conformación cromosómica para detectar interacciones de cromatina de largo alcance mediadas por una proteína de interés; enChIP, una técnica que emplea el sistema CRISPR/Cas9 para apuntar a regiones genómicas específicas y RIP-ChIP/RIP-Seq, una modificación de ChIP utilizada para analizar las interacciones proteína-ARN.

Comparación entre N-ChIP y X-ChIP

N-ChIP y X-ChIP tienen sus propias ventajas y desventajas. N-ChIP da como resultado una unión de anticuerpos más fuerte y una inmunoprecipitación eficiente y altamente específica. Sin embargo, N-ChIP sólo es adecuado en el caso de proteínas estrechamente unidas, como las histonas, ya que los factores de transcripción pueden desprenderse durante el procesamiento. Además, no toda la cromatina digerida por nucleasa se solubiliza, lo que provoca que falten ciertas fracciones de la muestra. X-ChIP es una metodología excelente para estudiar factores de transcripción que no están estrechamente unidos al ADN debido a su paso de entrecruzamiento. El ensayo X-ChIP también es más sensible que N-ChIP y requiere menores cantidades de muestras y anticuerpos. Las desventajas de X-chip incluyen posible dificultad en la fragmentación debido al exceso de entrecruzamiento y falsos positivos debido al entrecruzamiento de interacciones transitorias entre proteínas y ADN.

Inmunoprecipitación de cromatina: ChIP

La inmunoprecipitación de cromatina, o ChIP, es una técnica basada en anticuerpos que se utiliza para identificar sitios en el ADN que se unen a factores de transcripción de interés o ...

Education

Educación

JoVE Core

Molecular Biology

Biología Molecular

Unit 2

Unidad 2

Analyzing Gene Expression and Function

Análisis de la expresión y función génica

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

chip methods for dna-protein binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation.

ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes are then isolated by immunoprecipitation with specific antibodies. N-ChIP does not use cross-linking. Instead, it uses nucleases to digest the chromatin.

After the DNA of interest is isolated, it can be analyzed in several ways. PCR, microarrays, and Southern blot can be used to study the captured DNA. The sample can also be sent for deep sequencing, which is called ChIP-Seq.

ChIP can also be adapted for other kinds of analysis. ChIA-PET combines ChIP with chromosome conformation capture to detect long-range chromatin interactions that are mediated by a protein of interest. enChIP uses the CRISPR/Cas9 system to target specific genomic regions. RIP-ChIP and RIP-Seq are used to study protein-RNA interactions.

N-ChIP and X-ChIP each have strengths and limits. N-ChIP gives stronger antibody binding and efficient, highly specific immunoprecipitation. It works best with tightly bound proteins such as histones, but transcription factors may be lost during processing. Some nuclease-digested chromatin may also remain insoluble, so parts of the sample can be missed.

X-ChIP is useful for studying transcription factors that do not bind tightly to DNA because it includes a cross-linking step. It is also more sensitive than N-ChIP and uses less sample and fewer antibodies. Its drawbacks include possible problems with fragmentation if cross-linking is too strong and false positives from transient DNA-protein interactions.

ChIP Methods for DNA-Protein Binding

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Explore how ChIP detects DNA-protein binding, compares X-ChIP and N-ChIP, and shows how captured DNA can be analyzed by PCR, sequencing, and more.

ChIP Methods for DNA-Protein Binding | JoVE K12

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BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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