Biz, bu video ve makale eleştirmeyi katılımları için Brown ve Lam laboratuvarları üyelerine teşekkür etmek istiyorum. Bu çalışma, Kanada Sağlık Araştırma ve Michael Smith Sağlık Araştırma Vakfı Enstitüsü&#39;nün fonları tarafından desteklenmiştir.

DNA EKSTRAKSİYON VE ÖRNEK HAZIRLAMA Farklı örnekleri çeşitli DNA (kültür hücreleri, taze yanı formalin ile fikse parafine gömülü dokularda dondurulmuş) MeDIP için kullanılabilir. Yüksek derecede saflaştırılmış DNA, histon gibi ilişkili proteinlerin olmadan kullanmak önemlidir. DNA kantitatif ve antikor bağlanma ile müdahale örnek olarak, mümkün olduğunca çok RNA kaldırmak için de önemlidir. MeDIP için kullanılan DNA miktar 200 ng kullanılabilir DNA miktarına bağlı olarak 1 mg arasında olabilir. Bu protokol göstermek için, 1 mg DNA kullanılır. Aşağıdaki protokol kültürlü hücreler yüksek kaliteli çift iplikli DNA sağlayacaktır. Diğer protokoller, diğer örnek türlerinin DNA ekstraksiyonu için istihdam edilmelidir. <ol><li> 1.7 ml Eppendorf tüp bir hücre pelet sindirim tampon 400 ul ekleyin. </li><li> Tüp proteinaz K 100 mikrogram ve 50 gece inkübe ° C </li><li> 500 ul fenol pH 7 ekleyin ve alt üst edilerek yavaşça ama iyice karıştırın. </li><li> Oda sıcaklığında 10 dakika için 13 000 g Spin. </li><li> Yeni bir tüp sulu (üst) kesir çıkarın. </li><li> Tekrar bir kez 3 ile 5 arasındaki adımları. </li><li> 500 ul 01:01 fenol / kloroform pH 7 ekleyin ve alt üst edilerek yavaşça ama iyice karıştırın. </li><li> Oda sıcaklığında 10 dakika için 13 000 g Spin. </li><li> Yeni bir tüp sulu (üst) kesir çıkarın. </li><li> RNaz A 40 mikrogram ekleyin ve 37 az 1 saat inkübe ° C. </li><li> 500 ul fenol pH 7 ekleyin ve alt üst edilerek yavaşça ama iyice karıştırın. </li><li> Oda sıcaklığında 10 dakika için 13 000 g Spin. </li><li> Yeni bir tüp sulu (üst) kesir çıkarın. </li><li> Tekrar bir kez, 13 üzerinden 11 Adımlar. </li><li> 500 ul 01:01 fenol / kloroform pH 7 ekleyin ve alt üst edilerek yavaşça ama iyice karıştırın. </li><li> Oda sıcaklığında 10 dakika için 13 000 g Spin. </li><li> Yeni bir tüp sulu (üst) kesir çıkarın. </li><li> 1/10th hacim 3 M sodyum asetat (40 ul) ekleyin ve iyice karıştırın. </li><li> % 100 etanol 2 cilt (900 ul) -20 ° C&#39;de, 20 dakika boyunca iyice karıştırın ve yer. </li><li> 20 dakika için 13 000 gr az Spin 4 ° C </li><li> , Etanol çıkarın darbe spin ve artık etanol çıkarın. </li><li> Yıkamak için 500 ul soğuk% 70 etanol ekleyin. 20 dakika için 13 000 gr az Spin 4 ° C </li><li> % 70 etanol çıkarın darbe spin ve pipetleme kalan etanol kaldırmak. </li><li> Hava pelet kalan etanol tüm izlerini silmek için oda sıcaklığında 10 dakika açık kapak ile kurulayın. </li><li> 4 gecede sterilize dH2O 50 ul yeniden süspanse DNA ° C </li><li> NanoDrop Spektrofotometre ile DNA kantitatif olarak tanımlanmaktadır. A 260: 1.8 280 oranı idealdir. </li><li> 100 baz puan (bp) merdiven ile bir agaroz jel üzerinde DNA kalitesi ve boyut aralığı belirleyin. SYBR Altın gibi DNA az miktarda son derece duyarlı bir boya ile boyanarak az 10 ng genomik DNA,% 1.7 agaroz jel üzerinde çalıştırılabilir. </li><li> Başına bir örnek silikonlu tüp, steril dH 2 O hacmi kalan 50 ul toplam hacmi 1 mg DNA hazırlamak </li></ol> DNA Sonikasyon DNA sonication ve MeDIP protokol tüp duvarları olmayan belirli bağlayıcı proteinlerin önlemek için siliconzied tüpler yapılmalıdır. DNA örnekleri için en uygun sonication kez jel elektroforezi (Adım 27) belirlenen DNA örneği parçalanma derecesine dayanır. Örneğin, kültürlü hücrelerinden çıkarılan DNA, çok yüksek molekül ağırlığı olmalı ve daha sonra genellikle kısmen bozulmuş arşiv örneklerden çıkarılan DNA daha fazla sonication gerekecektir. Örnekleri düzgün yüksek molekül ağırlığı varsa, tek tek her bir numune kontrol etmeden bu protokolü olarak açıklanan sonication ile devam etmek için bu noktada mantıklı. Örnekleri arşiv örnekleri ile parçalanmış iseniz, 300 100bp parçaları elde etmek için sonication kez azaltarak muhtemel sonication prosedürü uyum için gerekli olacaktır. Kısmen bozulmuş, sonication parametreleri optimizasyonu ilgi temsil eden bir örneklem üzerinde yapılan numunelerde süreci bekliyoruz. Jel elektroforezi (Adım 27) gözlenen DNA parçalanma derecesine göre, deneyci örnek için en uygun sonication zaman önceden hesaplamak. Burada yüksek molekül ağırlıklı DNA kullanılarak otomatik bir sonicating cihazı (Diagenode gelen Bioruptor, UCD-200 TM) ile sonication 300-1000 bp DNA parçalarının elde etmek için bir yöntem açıklanmaktadır. <ol><li> Biorupter içinde su, 4 ° C olması gerekir </li><li> Otomatik ayarları 7 dakika (30 sn 30 sn maksimum güç kapalı) sonikasyon. </li><li> Silikonlu 1.7 ml santrifüj tüpüne immunoprecipitation (IP) reaksiyonu için sonicated ürün ve yeri (40 ul) 800 ng çıkarın. </li><li> Girdi olarak hizmet kalan 200 ng (10 ul) () (4 mağaza ° C) referans DNA kenara koyun. </li&gt; </ol> Metillenmiş DNA OF IMMUNOPRECIPITATON <ol><li> 95 ° C&#39;de su banyosunda 10 dakika süreyle IP reaksiyonu (800 ng) için kullanılacak olan DNA denatüre. </li><li> Hemen buz üzerinde soğutun. Sonraki adıma geçmeden önce (yaklaşık 5 dakika buz üzerinde) tamamen DNA soğumasını bekleyin. </li><li> 5 mikrogram monoklonal antikor ekleyin. </li><li> IP tampon (Bu protokol boyunca oda sıcaklığında) 500 ul nihai hacmi ekleyin. </li><li> Tüp tutucu dönen 2 saat 4 ° C&#39;de inkübe edin. </li><li> 5. adımı tamamlandıktan hemen önce, yıkama (Adımlar 6-11) Dynabeads hazırlar. Öncelikle, iyice karıştırın tarafından flakon boncuk tekrar süspansiyon haline getirin. </li><li> Yeni silikonlu bir tüp içine (örneğin, 8 reaksiyon yaparsa, 9, yani 270 ul için yeterli boncuk kaldırmak), 30 ul reaksiyon başına yeniden süspanse Dynabeads artı 1 (~ 2 x 10 7) aktarın. </li><li> Oda sıcaklığında 2 dakika manyetik raf tüp yerleştirin. </li><li> Süpernatantı Pipet. Süpernatant çıkarırken, pipet ucu ile iç duvar (mıknatıs boncuk çekmek) karşı boncuk dokunmaktan kaçının. </li><li> Mıknatıs tüp çıkarın, boncuklar ve IP tampon aşırı miktarda (750-1000 ul ul) tekrar süspansiyon haline getirin. Oda sıcaklığında 2 dakika geri manyetik raf tüp yerleştirin. </li><li> Yıkama bir kez daha tekrarlayın ve sonra Adım 7 kaldırıldı orijinal hacminin IP tampon yıkanmış boncuklar tekrar süspansiyon haline getirin. </li><li> Her IP reaksiyon yıkanmış Dynabeads 30 ul ekle </li><li> 2 saat için dönen bir tüp tutucu inkübe 4 ° C </li><li> Inkübasyon tamamlandıktan sonra, oda sıcaklığında 2 dakika manyetik rafa yerleştirilir. </li><li> Süpernatantı Pipet. Pipet ucu ile tüpün iç duvarına (mıknatıs boncuk çekmek) dokunmaktan kaçının. IP tampon 500 ul ekleyin. Tüp içindekileri karıştırın ve 2 dakika için manyetik rafa geri koymak. 500 ul IP tampon bir kez yıkayın tekrarlayın. </li><li> Son yıkama süpernatantı çıkardıktan sonra, sindirim tampon 400 ul boncuklar tekrar süspansiyon haline getirin. </li><li> Proteinaz K 100 mikrogram ile reaksiyon davranın ve 50 gece inkübe ° C </li></ol> IMMUNOPRECIPITATED DNA SAFLAŞTIRMA <ol><li> 500 ul 01:01 fenol / kloroform pH 7 ve iyice vorteks ekleyin. </li><li> Oda sıcaklığında 10 dakika için 13 000 g Spin. </li><li> Yeni bir tüp sulu (üst) kesir çıkarın. </li><li> Sulu ve organik katmanları arasında interfaz bulutlu görünürse, 1 ile 3 arasındaki adımları tekrarlayın. </li><li> 1 / 10 hacim 3 M sodyum asetat (40 ul) ve vorteks ekleyin. </li><li> 1 glikojen ul (20 mg / ml) ve vorteks ekleyin. </li><li> % 100 etanol, girdap ve -20 ° C&#39;de 20 dakika yerine 2 cilt (1000 ul) ekleyin. </li><li> 20 dakika için 13 000 gr az Spin 4 ° C </li><li> , Etanol çıkarın darbe spin ve artık etanol çıkarın. </li><li> Yıkamak için 500 ul soğuk% 70 etanol ekleyin. 4. 20 dakika boyunca 13 000 g Vortex kısaca, ve spin ° C </li><li> % 70 etanol çıkarın darbe spin ve pipetleme kalan etanol kaldırmak. </li><li> Hava pelet kalan etanol tüm izlerini silmek için oda sıcaklığında 10 dakika açık kapak ile kurulayın. </li><li> 10 ul yeniden süspanse DNA pelet dH 2 0 sterilize edilmelidir. </li></ol> PCR ile doğrulama <ol><li> MeDIP prosedürü kullanarak, normal bir insan DNA (erkek veya kadın) MeDIP protokol yaparak çalışma, metilasyon için zenginleştirilmiş olarak bilinen bir bölge ve daha sonra assaying olduğundan emin olmak için test edebilir. </li><li> PCR tüp MeDIP ürünün% 30, ve başka bir PCR tüpüne, MeDIP ürünün başka bir% 30 çıkarın. </li><li> DNA&#39;nın PCR tüp içine 10 ng ve 10 ng DNA&#39;nın başka bir PCR tüpünün içine koyun. Artık dört ayrı PCR reaksiyonları kurmak olmalıdır. </li><li> Tablo 1&#39;de belirtildiği gibi PCR H19 ve CTRL primerleri kullanarak gerçekleştirin. </li><li> Tablo 2&#39;de belirtildiği gibi 12.5 ul toplam hacmi ile PCR reaksiyonlar için iki mastermixes (her astar kümesi için biri) hazırlayın. </li><li> Thermocycle PCR Tablo 3&#39;te belirtilen şartlara. </li><li> PCR ürünlerinin görünüm için% 2&#39;lik agaroz jel üzerinde 5 ul çalıştırın. </li><li> Başarılı MeDIP için beklenen sonuçlar Tablo 4&#39;te gösterilmiştir. </li></ol> Tablolar Tablo 1: H19 ve PCR doğrulama için CTRL primerleri. <table border="1"><tbody><tr><td> Astar Seti </td><td> İleri Astar </td><td> Ters Astar </td><td> Beklenen Ürün Boyutu </td></tr><tr><td> H19 </td><td> H19_F 5&#39;-cgagtgtgcgtgagtgtgag </td><td> H19_R 5&#39;-ggcgtaatggaatgcttgaa </td><td> 174 bp </td></tr><tr><td> CTRL (kontrol) </td><td> CTRL_F5&#39;-gagagcattagggcagacaaa </td><td> CTRL_R 5&#39;-gttcctcagacagccacattt </td><td> 139 bp </td></tr></tbody></table> Tablo 2. PCR reaksiyonları için Mastermixes. <table border="1"><tbody><tr><td></td><td> H19 Mix (kişi başına RXN) </td><td> CTRL Mix (kişi başına RXN) </td></tr><tr><td&gt; GKD 2 O </td><td> 6,875 </td><td> 6,875 </td></tr><tr><td> 10X Tampon </td><td> 1,25 </td><td> 1,25 </td></tr><tr><td> dNTP karışımı (her biri 10 mM) </td><td> 0,25 </td><td> 0,25 </td></tr><tr><td> MgCl 2 (50 mM) </td><td> 0,25 </td><td> 0,25 </td></tr><tr><td> Astarlar (H19_F / R veya CTRL_F / R) (10 mcM her F / R) </td><td> 0,625 </td><td> 0,625 </td></tr><tr><td> Platinum Taq </td><td> 0,25 </td><td> 0,25 </td></tr></tbody></table> Tablo 3. PCR thermocyling. <table border="1"><tbody><tr><td></td><td> 95 ° C </td><td> 05:00 </td></tr><tr><td rowspan="3"> 40X </td><td> 95 ° C </td><td> 00:30 </td></tr><tr><td> 56 ° C </td><td> 00:30 </td></tr><tr><td> 72 ° C </td><td> 00:15 </td></tr></tbody></table> Tablo 4. Beklenen başarılı MeDIP PCR sonuçları. <table border="1"><tbody><tr><td></td><td colspan="2" align="center"> DNA </td></tr><tr><td> Primer kullanılan </td><td> IN </td><td> IP </td></tr><tr><td> H19 </td><td> Olumlu </td><td> Olumlu </td></tr><tr><td> CTRL </td><td> Olumlu </td><td> Negatif </td></tr></tbody></table>

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Hastalığında önemli rol DNA metilasyon oynar büyüyen bir farkındalık vardır, bu nedenle testlerin geliştirilmesi, bu değişikliği ölçmek için 13, 12, 3 giderek daha önemli hale gelmektedir. MeDIP teknik tüm genomu ve lokus spesifik seviyesi 6, 7, tarama için bir mükellef araçtır . Bu teknik, DNA başlayan sınırlı miktarda kullanarak DNA metilasyon seviyelerini hızlı bir görünüm sağlar ve farklı kaynaklardan arasında kolay karşılaştırma için sağlar. MeDIP ürün...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Biorupter sonicator</td>
<td>Tool</td>
<td>Diagenode</td>
<td>UCD-200 TM</td>
<td>&nbsp;</td>
<tr>
<td>1.7ml SafeSeal Microcentrifuge Tubes</td>
<td>Other</td>
<td>Sorenson BioScience</td>
<td>11510</td>
<td>&nbsp;</td>
<tr>
<td>ND 3300 Spectrophotometer</td>
<td>Tool</td>
<td>NanoDrop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibody: Anti-5'-methylcytosine mouse mAb</td>
<td>Reagent</td>
<td>CalBiochem</td>
<td>162 33 D3</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>112-01D</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Tube Rack</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>CS15000</td>
<td>&nbsp;</td>
<tr>
<td>Mini LabRoller</td>
<td>Tool</td>
<td>Labnet International</td>
<td>H5500</td>
<td>&nbsp;</td>
<tr>
<td>IP Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td>
</tr>
<tr>
<td>Digestion Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td>
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methylated dna immunoprecipitation

DNA metilasyon paternlerinin belirlenmesi epigenetik çalışma metilasyon gen ekspresyonu üzerinde önemli etkileri olduğu bilindiği gibi, ortak bir prosedür ve hastalığın yanı sıra normal gelişimi ile ilgili<sup&gt; 1-4</sup&gt;. Böylece, metil DNA ve non-metil DNA arasında ayrım yapma yeteneği, bu tür çalışmalar için metilasyon profilleri üreten için gerekli. Metillenmiş DNA immunoprecipitation (MeDIP) ilgi bir örnek metillenmiş DNA çıkarılması için etkin bir tekniktir<sup&gt; 5-7</sup&gt;. DNA az 200 ng örnek bir antikor ya da immunoprecipitation (IP), reaksiyon için yeterli. DNA boyutu 300-1000 bp arasında değişen parçaları içine sonicated ve immunoprecipitated (IP) ve giriş (IN) kısımları ayrılır. IP DNA daha sonra, monoklonal antikor metillenmiş DNA bağlamak için izin denatüre ve sonra anti-5&#39;mC inkübe ısı. Bundan sonra, primer antikor afinite ile ikincil bir antikor içeren manyetik boncuk eklenir ve inkübe edilir. Bu boncuk bağlanan antikorlar, ilk adımda kullanılan monoklonal antikor bağlanacaktır. Antikor kompleksi (metil DNA) bağlı DNA çözüm kompleksleri dışarı çekmek için bir mıknatıs kullanarak DNA geri kalanından ayrılır. IP tampon kullanarak birçok yıkar sonra ilişkisiz, non-metil DNA kaldırmak için yapılır. Metillenmiş DNA / antikor komplekslerini sonra sadece metillenmiş DNA zedelemeden antikorlar sindirmek için Proteinaz K ile sindirilir. Protein madde kaldırmak için kloroform ekstraksiyon ve daha sonra daha sonra kullanmak üzere çöktürülmüş ve su yeniden süspanse: fenol ile zenginleştirilmiş DNA arındırılır. IP amplifikasyon ürünleri analiz ederek ve eksikliği bilinen ve metil dizileri içerdiği bilinen bölgeler için DNA&#39;nın MeDIP prosedürün etkinliğini doğrulamak için PCR teknikleri kullanılabilir. Saflaştırılmış metillenmiş DNA lokus spesifik (PCR) veya genom (mikroarray ve sıralama) metilasyon çalışmaları için kullanılan ve gen ekspresyon profili ve dizi karşılaştırmalı genom hibridizasyonu gibi diğer araştırma araçları ile birlikte uygulandığında (özellikle yararlıdır CGH)<sup&gt; 8</sup&gt;. DNA metilasyon içine daha fazla araştırma da, aberrantly metillenmiş DNA ile karakterize olan kanser gibi hastalıklar için yeni tedavi veya prognostik araştırma araçları geliştirilmesinde yararlı olabilir, yeni epigenetik hedefler, keşfine yol açacaktır<sup&gt; 2, 4, 9-11</sup&gt;.

Watch this Scientific Journal Video about Methylated DNA Immunoprecipitation at JoVE.com

Methylated DNA Immunoprecipitation

Bu video metillenmiş DNA immunoprecipitation (MeDIP) için protokol göstermektedir. MeDIP seçici (anti-5 mC) 5-methylcytosine özgüllüğü antikorlar kullanılarak genomik DNA örneği metillenmiş DNA parçaları ayıklar iki günlük bir işlemdir.

Metillenmiş DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Research

JoVE Journal

Biology

23.2K Görüntüleme.  BC Cancer Research Centre. Bu video metillenmiş DNA immunoprecipitation (MeDIP) için protokol göstermektedir. MeDIP seçici (anti-5 mC) 5-methylcytosine özgüllüğü antikorlar kullanılarak genomik DNA örneği metillenmiş DNA parçaları ayıklar iki günlük bir işlemdir.

Video: Methylated DNA Immunoprecipitation

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biyoloji

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikor Profil Lusiferaz Immunoprecipitation Sistemleri (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA Kararlı-İzotop (DNA-SIP) Probing

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP FCM: Sitometrisi ile Algılandı Immunoprecipitation

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Erken evre Fare Embriyolar kullanarak Doku özel Genler Kromatin Immunoprecipitation Testi

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Kullanarak Kromatin immünopresipitasyon (ChIP) Drosophila Doku

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

SeqA-DNA kompleksleri kristalleşmesi için DNA dupleksler optimizasyonu İteratif

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Biyokütle Limit Tutarlar kullanarak Verimli Kromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...