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

Immunoprecypitacja chromatyny, w skrócie ChIP, jest techniką badania interakcji białko-DNA, które regulują ekspresję genów.
U eukariontów DNA jest owinięte wokół białek histonowych i tworzy kompleks znany jako nukleosom, który dalej grupuje się w strukturę znaną jako chromatyna, aby pomóc w ciasnym upakowaniu DNA w komórkach.
Ekspresja genów jest regulowana poprzez modyfikacje histonów, które rozwijają lub napinają te struktury, a także białka, które wiążą się z regionami cis-regulatorowymi na DNA.
W ChIP chromatyna jest rozkładana, a przeciwciała, które wiążą się z modyfikacjami histonów lub białkami regulatorowymi, są wykorzystywane do izolowania docelowych cząsteczek z powiązanym DNA.
Pierwszym krokiem w tym procesie jest sieciowanie białka z DNA za pomocą środka sieciującego, takiego jak formaldehyd. Powoduje to unieruchomienie białka na DNA, oznaczając miejsce wiązania białka. Po usieciowaniu chromatyna jest mechanicznie ścinana na krótkie fragmenty o długości od 100 do 200 par zasad. Ten proces jest znany jako X-ChIP.
Alternatywną metodą jest N-ChIP, w której nukleazy bezpośrednio trawią DNA z chromatyny na krótkie fragmenty, bez wcześniejszego sieciowania.
Immunoprecypitacja jest przeprowadzana poprzez wprowadzenie przeciwciał skierowanych przeciwko białkom regulatorowym do roztworu zawierającego ścięte lub strawione DNA. Przeciwciała te są powiązane ze środkami, które pomagają w selektywnej izolacji.
Jedną z powszechnych metod jest łączenie przeciwciał z kulkami magnetycznymi. Magnes służy do izolowania przeciwciał wraz z wszelkimi związanymi cząsteczkami.
Kompleks jest płukany w celu zmycia wszelkich luźno związanych zanieczyszczeń. Docelowe cząsteczki są odłączane za pomocą detergentu, takiego jak SDS.
W przypadku X-ChiP sieciowanie jest odwracane za pomocą podwyższonych temperatur, a związane z nim regulatory lub histony są degradowane za pomocą proteaz, pozostawiając DNA. 
Sekwencjonowanie powiązanego DNA może pomóc w identyfikacji sekwencji cis-regulatorowej, z którą związane było białko będące przedmiotem zainteresowania, lub genu, którego ekspresja jest modulowana przez określoną modyfikację histonów.

<ol>
	<li>Gade, P., & Kalvakolanu, D. V. (2012). Test immunoprecypitacji chromatyny jako narzędzie do analizy aktywności czynnika transkrypcyjnego. Metody w biologii molekularnej (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). Protokół metody szybkiej immunoprecypitacji chromatyny (ChIP). Protokoły natury, 1(1), 179–185. doi:10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004). Test immunoprecypitacji chromatyny. Biotechniki, 37(6), 961-969.</li>
</ol>

immunoprecypitacja chromatyny – chip

Immunoprecypitacja chromatyny (ChIP) to technika oparta na przeciwciałach, stosowana do identyfikacji miejsc w DNA, które wiążą się z interesującymi czynnikami transkrypcyjnymi lub białkami histonowymi. Pomaga także określić rodzaj modyfikacji histonów, takich jak acetylacja, fosforylacja lub metylacja. <h4>Rodzaje ChIP</h4> ChIP można podzielić na dwa typy - X-ChIP i N-ChIP. X-ChIP obejmuje sieciowanie in vivo histonów i białek regulatorowych z DNA, fragmentację DNA przez sonikację i izolację kompleksów białko-DNA poprzez immunoprecypitację ze specyficznymi przeciwciałami. Z drugiej strony N-ChIP nie polega na sieciowaniu DNA z białkami, a trawienie odbywa się za pomocą nukleaz. <h4>Dalsza analiza interakcji DNA-białko</h4> Po wyizolowaniu interesującego DNA do analizy można zastosować techniki takie jak PCR, mikromacierze lub Southern blot. Alternatywnie, ten DNA można zastosować do głębokiego sekwencjonowania, znanego jako ChIP-Seq. ChIP można modyfikować przy użyciu innych metodologii do różnych analiz, takich jak ChIA-PET, technika łącząca zasady ChIP z wychwytywaniem konformacji chromosomu w celu wykrywania interakcji chromatyny dalekiego zasięgu, w których pośredniczy białko będące przedmiotem zainteresowania; enChIP, technika wykorzystująca system CRISPR/Cas9 do celowania w określone regiony genomu oraz RIP-ChIP/RIP-Seq, modyfikacja ChIP stosowana do analizy interakcji białko-RNA. <h4>Porównanie N-ChIP i X-ChIP</h4> N-ChIP i X-ChIP mają swoje zalety i wady. N-ChIP powoduje silniejsze wiązanie przeciwciał oraz wydajną i wysoce specyficzną immunoprecypitację. Jednakże N-ChIP jest odpowiedni tylko w przypadku ściśle związanych białek, takich jak histony, ponieważ czynniki transkrypcyjne mogą zostać odłączone podczas przetwarzania. Ponadto nie cała chromatyna trawiona nukleazą ulega rozpuszczeniu, co powoduje brak niektórych frakcji próbki. X-ChIP to doskonała metodologia badania czynników transkrypcyjnych, które nie są ściśle związane z DNA ze względu na etap sieciowania. Test X-ChIP jest również bardziej czuły niż N-ChIP i wymaga mniejszych ilości próbek oraz przeciwciał. Wady X-chip obejmują możliwą trudność w fragmentacji z powodu nadmiernego usieciowania i fałszywie dodatnie wyniki z powodu usieciowania przejściowych interakcji białko-DNA.

Immunoprecypitacja chromatyny – ChIP

Immunoprecypitacja chromatyny lub ChIP to technika oparta na przeciwciałach stosowana do identyfikacji miejsc na DNA, które wiążą się z interesującymi czynnikami transkrypcyjnymi lub ...

Education

JoVE Core

Molecular Biology

Biologia molekularna

Unit 2

Jednostka 2

Analyzing Gene Expression and Function

Analiza ekspresji i funkcji genów

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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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