Name: chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells
Uploaded: 2010-07-29T17:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Bu çalışma, Virginia Üniversitesi, Virginia Üniversitesi Kanser Merkezi ve Thomas F. ve Kate Miller Jeffress Memorial Trust tarafından desteklenmektedir. James E. Darnell Lab (Rockefeller Üniversitesi) Robert Ross Lab (Fordham Üniversitesi), hücre hatları için teşekkür ederiz.

PLANLAMA ChIP deney planlanan gün önce, hücrelerin yeterli sayıda kullanılabilir, böylece kültüre olmalıdır. Her ChIP saflık ~ 1-1.5 x 10 7 hücre gerektirdiğini varsayalım. Her zaman negatif (IgG) ve pozitif (pan histon antikor) denetimleri ve yinelenen deneyler yapmak için plan. İpucu - 2FTGH hücreler için, her ChIP tahlil% 80 confluency yaklaşık 15 cm doku kültürü çanak gerektirir. BÖLÜM 1: Kromatin HAZIR...

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Ana hatlarıyla ChIP protokol laboratuarda tahlil yirmiden fazla farklı histon modifikasyonları başarıyla kullanılmaktadır. Buna ek olarak, transkripsiyon faktörleri doluluk değişiklikler karakterize, histon değiştirerek birkaç IFN-γ kaynaklı genlerin, histon modifikasyonları ve RNA Polimeraz II transkripsiyon makine kabul enzimler, proteinler. Ayrıca, kanser kök hücre 10 farklılaşması sırasında histon modifikasyonları değişiklikleri karakterize etmek için prosedürü kullandık . <...

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		<th>Comment</th>
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<td>37% Formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-500</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform/Isoamyl alcohol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>AC32715-5000</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitors</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1836153</td>
<td>&nbsp;</td>
<tr>
<td>Cosmic Calf Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30087.04N</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30243</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D0632</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Pharmco</td>
<td>zp1110EP</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>901393</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
<tr>
<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
<td>&nbsp;</td>
<tr>
<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LiCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4408</td>
<td>&nbsp;</td>
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<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
<td>&nbsp;</td>
<tr>
<td>Phenol/chloroform/isoamyl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500130</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein A Agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein G Agaorse</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-201</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP166-500</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5941</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-K36me3</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab9050</td>
<td>&nbsp;</td>
<tr>
<td>Anti-STAT1</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-345X</td>
<td>&nbsp;</td>
<tr>
<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
<td>&nbsp;</td>		</tbody>
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chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

STAT (sinyal dönüştürücü ve transkripsiyon aktivatörü) ekstrasellüler ligandların çeşitli yanıt olarak, transkripsiyon faktörleri hızla onlar tek bir tirozin kalıntısı 1 fosforilasyon ile aktive olan hücre yüzey reseptörleri sitoplazmada gizli devlet işe alınırlar. Daha sonra hedef genlerin transkripsiyon sürücü, büyüme, farklılaşma, homeostazı ve immün yanıtı etkileyen çekirdeği dimerize ve yerini değiştirmek. Beklendiği gibi, normal hücre süreçlerini yaygın tutulum verilen, İST fonksiyonu disregülasyonu özellikle 2 ve otoimmün hastalıklar 3 kanserleri, insan hastalığına katkıda bulunur. Bu transkripsiyon 4,5 kromatin şablonunda değişiklik tarafından düzenlenir kurulmuştur. Bu değişiklikler, ATP-bağımlı komplekslerinin yanı sıra kovalent histon modifikasyonları ve DNA metilasyon 6 faaliyetleri içerir . STAT aktivasyon gen ekspresyonu hem hızlı ve hem de geçici olduğundan, İST-bağımlı gen lokuslar modüle kromatin şablon için özel mekanizmalar gerektirir. Bu mekanizmalar tanımlamak için, histon modifikasyonları ve enzimatik faaliyetleri STAT sinyalizasyon cevap gen lokuslar onları oluşturmak karakterize eder. Bu protokol kromatin immunoprecipitation, İST aktif gen ekspresyonu kromatin sinyalizasyon çalışması için değerli bir yöntem açıklanmıştır.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Bu protokol, kromatin immunoprecipitation (ChIP) bir sinyal iletim yolu ile indüklenen transkripsiyonu düzenleyen kromatin şablon dinamik değişiklikleri incelemek için nasıl kullanıldığını açıklar.

Kromatin Immunoprecipitation İnsan Hücrelerinde Aktif Gen İfadesi Testi Dinamik Histon Modifikasyon (ChIP)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

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