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

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Chromatin Immunoprecipitation- ChIP

Die Chromatin-Immunpräzipitation, abgekürzt ChIP, ist eine Technik zur Untersuchung der Protein-DNA-Wechselwirkungen, die die Genexpression regulieren.
In Eukaryoten ist die DNA um Histonproteine gewickelt und bildet einen Komplex, der als Nukleosom bekannt ist und sich weiter zu einer Struktur gruppiert, die als Chromatin bekannt ist, um die dichte Verpackung der DNA in den Zellen zu unterstützen.
Die Genexpression wird durch Histonmodifikationen reguliert, die diese Strukturen aufwickeln oder verengen, sowie durch Proteine, die mit cis-regulatorischen Regionen auf der DNA assoziiert sind.
Bei ChIP wird das Chromatin abgebaut und Antikörper, die an Histonmodifikationen oder regulatorische Proteine binden, werden verwendet, um die Zielmoleküle mit der zugehörigen DNA zu isolieren.
Der erste Schritt in diesem Prozess besteht darin, das Protein mit Hilfe eines Vernetzungsmittels wie Formaldehyd mit der DNA zu vernetzen, wodurch das Protein auf der DNA immobilisiert wird und die Bindungsstelle des Proteins markiert wird. Nach der Vernetzung wird das Chromatin mechanisch in kurze Fragmente von 100 bis 200 Basenpaaren geschert. Dieser Prozess wird als X-ChIP bezeichnet.
Eine alternative Methode ist N-ChIP, bei der Nukleasen DNA aus dem Chromatin direkt in kurze Fragmente verdauen, ohne vorherige Vernetzung.
Die Immunpräzipitation erfolgt durch die Einführung von Antikörpern, die auf die regulatorischen Proteine in der Lösung abzielen, die gescherte oder verdaute DNA enthält. Diese Antikörper sind mit Wirkstoffen verknüpft, die bei der selektiven Isolierung helfen.
Eine gängige Methode ist die Verknüpfung der Antikörper mit magnetischen Kügelchen. Ein Magnet wird verwendet, um die Antikörper zusammen mit den gebundenen Molekülen zu isolieren.
Der Komplex wird gespült, um lose verbundene Verunreinigungen abzuwaschen. Die Zielmoleküle werden mit Hilfe eines Reinigungsmittels, wie z. B. SDS, abgelöst.
Im Falle von X-ChiP wird die Vernetzung mit Hilfe erhöhter Temperaturen rückgängig gemacht und die zugehörigen Regulatoren oder Histone werden mit Hilfe von Proteasen abgebaut, wobei die DNA zurückbleibt. 
Die Sequenzierung der assoziierten DNA kann helfen, die cis-regulatorische Sequenz zu identifizieren, an die das Protein von Interesse gebunden war, oder das Gen, dessen Expression durch eine bestimmte Histonmodifikation moduliert wird.

<ol>
	<li>Gade, P., & Kalvakolanu, D. V. (2012). Chromatin-Immunpräzipitationsassay als Werkzeug zur Analyse der Aktivität von Transkriptionsfaktoren. Methoden der Molekularbiologie (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). Protokoll für die Methode der schnellen Chromatin-Immunpräzipitation (ChIP). Naturprotokolle, 1(1), 179–185. doi:10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004). Chromatin-Immunpräzipitations-Assay. Biotechniken, 37(6), 961-969.</li>
</ol>

chromatin-immunpräzipitation – chip

Die Chromatin-Immunpräzipitation, oder ChIP, ist eine antikörperbasierte Technik, die verwendet wird, um Stellen auf der DNA zu identifizieren, an die Transkriptionsfaktoren von Interesse oder Histonproteine binden. Sie hilft auch, die Art der Histonmodifikationen wie Acetylierung, Phosphorylierung oder Methylierung zu bestimmen.

Arten von ChIP

ChIP kann in zwei Typen unterteilt werden - X-ChIP und N-ChIP. X-ChIP beinhaltet das in vivo Vernetzen von Histonen und regulatorischen Proteinen mit der DNA, das Fragmentieren der DNA durch Sonikation und das Isolieren der Protein-DNA-Komplexe durch Immunpräzipitation mit spezifischen Antikörpern. Im Gegensatz dazu beinhaltet N-ChIP kein Vernetzen von DNA mit Proteinen, und die Verdauung erfolgt mittels Nukleasen.

Nachgelagerte Analyse von DNA-Protein-Interaktionen

Nach der Isolierung der interessierenden DNA können Techniken wie PCR, Mikroarrays oder Southern-Blot zur Analyse verwendet werden. Alternativ kann diese DNA für die Tiefensequenzierung verwendet werden, bekannt als ChIP-Seq. ChIP kann durch andere Methoden für verschiedene Analysen modifiziert werden, wie ChIA-PET, eine Technik, die die Prinzipien von ChIP mit der Chromosomenkonformations-Erfassung kombiniert, um langreichweitige Chromatin-Interaktionen zu erkennen, die über ein Protein von Interesse vermittelt werden; enChIP, eine Technik, die das CRISPR/Cas9-System verwendet, um spezifische genomische Regionen zu zielen; und RIP-ChIP/RIP-Seq, eine Modifikation von ChIP, die verwendet wird, um Protein-RNA-Interaktionen zu analysieren.

Vergleich zwischen N-ChIP und X-ChIP

N-ChIP und X-ChIP haben ihre eigenen Vor- und Nachteile. N-ChIP führt zu einer stärkeren Antikörperbindung und einer effizienten und hochspezifischen Immunpräzipitation. Allerdings eignet sich N-ChIP nur, wenn die Proteine, wie Histone, fest gebunden sind, da Transkriptionsfaktoren während der Verarbeitung abgelöst werden können. Zusätzlich wird nicht das gesamte nuklease-verdaute Chromatin gelöst, was dazu führt, dass bestimmte Fraktionen der Probe fehlen. X-ChIP ist eine hervorragende Methodik für die Untersuchung von Transkriptionsfaktoren, die nicht fest an die DNA gebunden sind, dank des Vernetzungsschritts. Der X-ChIP-Assay ist auch empfindlicher als N-ChIP und benötigt geringere Mengen an Proben sowie Antikörpern. Nachteile von X-ChIP umfassen mögliche Schwierigkeiten bei der Fragmentierung aufgrund übermäßiger Vernetzung und falsch positive Ergebnisse durch die Vernetzung von transienten DNA-Protein-Interaktionen.

Chromatin-Immunpräzipitation – ChIP

Die Chromatin-Immunpräzipitation (ChIP) ist eine antikörperbasierte Technik, die zur Identifizierung von Stellen auf der DNA verwendet wird, die an Transkriptionsfaktoren von Interesse binden oder ...

Education

Lehre

JoVE Core

Molecular Biology

Molekularbiologie

Unit 2

Einheit 2

Analyzing Gene Expression and Function

Analyse von Genexpression und -funktion

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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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key terms and concepts

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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