Bu çalışma, Alman Bilim Vakfı (DFG) ve Alman federal ve eyalet hükümetleri Mükemmellik Girişimi tarafından finanse edildi.

1. Bitki materyali, Crosslinking <ol><li> Hasat 1-2g Arabidopsis 50 ml plastik tüp yaprak (6, 10 cm rozet çapa kadar) ve crosslinking tampon ile 40ml kadar doldurmak. </li><li> Yaprakları, tampon yüzeyi üzerinde özel bir filtre süngeri sağ doldurma, örneğin dalmış muaftır emin olun. Sonra bir desikatöre tüpler yerleştirin. </li><li> Vakum 10dk sızmak. Crosslinking durdurmak için her tüp 2M glisin 2.5ml eklemek ve glisin eşit dağılımını garanti tüpler ters. </li><li> Vakum başka bir 5min sızmak ve bir elek üzerinde yaprakları hasat sırasında sıvı atın. </li><li> Yıkama 1L, sonra bir bardak beher su, kağıt havlu ile iyice kuru yapraklar ile iki kez bırakır. </li><li> Taze bir plastik tüp yaprakları toplayın ve sıvı nitrojen içinde dondurarak. Mağaza -80 ° C kadar kromatin izolasyon bırakır. </li></ol> 2. Kromatin izolasyonu <ol><li> Yapraklar kullanana kadar sıvı azot tutuldu harç ve dişilik organı ile zemin. </li><li> 50-ml plastik tüp toz transferi ve 30ml ekstraksiyon tamponu # 1 geçici olarak askıya. </li><li> Bir tepegöz çalkalayıcı 4 ° C&#39;de 15 dakika inkübe. </li><li> 50-ml taze bir plastik tüp içine dört kat miracloth boyunca Filtrat süspansiyon. </li><li> 2.800 xg 20min için Spin 4 ° C </li><li> Süpernatantı ve 1 ml tampon ekstraksiyon # 2 fırça ile pelet askıya. İlk tampon # 2 küçük bir hacim eklemek, sonra pelet bir boya fırçası ile askıya alma ve sonra tampon geri kalanını ekleyin. </li><li> 4 12,000 xg&#39;de 10 dakika süreyle bir 1.5mL mikrofuge&#39;ye tüp ve spin süspansiyon transfer ° C </li><li> Bu arada, 1.5mL ekstraksiyon tamponu # 3 içeren 2mL mikrofuge&#39;ye tüpleri hazırlayın. </li><li> Bükülmüş tüp (adım 2.7) süpernatantı ve ekstraksiyon tamponu # 3 300μL pelet askıya. İlk olarak, tampon # 3 küçük hacimli bir boya fırçası ile pelet askıya, ve sonra geri kalan tampon eklemek. </li><li> Dikkatle pipetlemeyin adım 2.8 &#39;de hazırlanan 1,5 mL tampon ekstraksiyon # 3 üst pelet (adım 2.9) askıya aldı. Iki faz karışımı olmayacak emin olun. 4 16.000 xg&#39;de 1s için Spin ° C. </li><li> Süpernatantı ve sonication tampon 300μL bir boya fırçası ile kromatin pelet askıya. </li><li> DNA boyutu yaklaşık 400bp Sonotrode ile ya da ultrasonik banyoda kromatin sonikasyon. Sonicating zaman, emin kromatin yaklaşık 30 üzerinde ısıtılır ° C ısıya duyarlı çapraz bağları korumak için olun. Sonication süresi farklı sonication cihazlar arasında önemli ölçüde farklılık gösterir, çünkü deneysel olarak belirlenir. Rehberlik için, iki farklı aygıtlar için ayarları verilmiştir: </li></ol> 0.6mL mikrofuge&#39;ye tüp Bandelin marka Sonotrode maruz kromatin% 25 güç, çevrim = 50 ayarları ile beş kez 20 patlamaları ile sonication. Bunu yaparken, bir buz banyosunda her 20 inci patlamaları sonra serin bir örnek . Diagenode Bioruptor ultrasonik banyo ile sonication için, su banyosu ve buz ile doldurun ve &#39;yüksek&#39; ayarı ile 1.5mL mikrofuge&#39;ye tüpler 30 saniye on kat sonikasyon. Her 30 saniye sonra, başka bir 30 saniye boyunca serin örnekleri. <ol start="13"><li> Spin 4 16.000 g 5min için örnek sonicated ° C çözünmemiş malzeme hızlandırabilir. Süpernatant doğrudan immunoprecipitation için kullanılabilir. Alternatif olarak, numuneler, sıvı nitrojen içinde dondurulmuş ve -80 ° C ye kadar daha fazla kullanım saklanan şok olabilir. </li></ol> 3. Immunoprecipitation <ol><li> 40μL protein-A antikor bağlayıcı tampon agaroz ve 1.8mL. Içeren 2 ml mikrofuge&#39;ye tüpleri hazırlayın Kromatin hazırlık 200μL (adım 2.13) ekleyin. </li><li> Havai bir çalkalamalı 1s tüpleri inkübe edin. </li><li> Protein-A agaroz boncuk atın ve immunoprecipitation için supernatant kullanabilirsiniz. </li><li> Kromatin konsantrasyonu (&#39;input&#39;) belirlemek için bir 40μL-kısım çıkarın. </li><li> 30μL protein-A agaroz ve 400μL sonicated kromatin süspansiyon özgü reaktifler ve teçhizat aşağıdaki tabloda belirtilen değişiklik-spesifik antikor miktarı. </li><li> Bir tepegöz çalkalayıcı 2.5h veya gece boyunca inkübe 4 ° C </li><li> 440 x g. 2min için boncuk aşağı Spin </li><li> Boncuk pelet 900μL (aşağıdaki listeye bakın) her yıkama tamponu ile yıkayın. Her tampon için, bir havai çalkalayıcı 4 ° C, 10 dakika süreyle tampon inkübe boncuk, sonra 440 xg&#39;de 2min dönmeye supernatant atın ve bir sonraki tampon eklemek. <ol><li> Düşük tuz yıkama tamponu </li><li> Yüksek tuz yıkama tamponu </li><li> LiCl yıkama tamponu </li><li> Te yıkama tamponu </li></ol></li><li> Son yıkamadan sonra, kalan herhangi bir tampon tamamen kaldırmak. </li></ol> 4. Histon ve DNA De-crosslinking ve çöktürülmüş DNA saflaştırılması <ol><li> Agaroz pelet ve 3.4 adım 40μl &#39;giriş&#39; örnek bir vorteks karıştırıcı 5min için karışımı, spin örnekleri ve incu de crosslinking tampon 100μL ekle65 ° C gecede kesilmek. </li><li> Ticari bir DNA veya PCR saflaştırma kiti ile DNA arındırın. </li><li> Genellikle son sütun eluat 01:05 seyreltme 5μl gerçekleştirmek için yeterli geleneksel lokus spesifik gerçek zamanlı kantitatif RT-PCR (RT-qPCR). </li></ol> 5. Tamponların Tablosu <table border="1"><tbody><tr><td colspan="2"> Crosslinking tampon </td></tr><tr><td> Sodyum bütirat </td><td> 10mM </td></tr><tr><td> Sakaroz </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 10mM </td></tr><tr><td> ß-mercaptoethanol </td><td> 5mM </td></tr><tr><td> Formaldehit </td><td> % 3 (v / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Ekstraksiyon tamponu # 1 </td></tr><tr><td> Sodyum bütirat </td><td> 10mM </td></tr><tr><td> Sakaroz </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 10mM </td></tr><tr><td> ß-mercaptoethanol </td><td> 5mM </td></tr><tr><td> Komple proteaz inhibitörü </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Ekstraksiyon tamponu # 2 </td></tr><tr><td> Sodyum bütirat </td><td> 10mM </td></tr><tr><td> Sakaroz </td><td> 250mm </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 10mM </td></tr><tr><td> ß-mercaptoethanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 10mM </td></tr><tr><td> Triton X-100 </td><td> % 1 (w / v) </td></tr><tr><td> Komple proteaz inhibitörü </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Ekstraksiyon tamponu # 3 </td></tr><tr><td> Sodyum bütirat </td><td> 10mM </td></tr><tr><td> Sakaroz </td><td> 1,7 </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 10mM </td></tr><tr><td> ß-mercaptoethanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 2mm </td></tr><tr><td> Triton X-100 </td><td> % 0,15 (w / v) </td></tr><tr><td> Komple proteaz inhibitörü </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Sonikasyon tampon </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 25mm </td></tr><tr><td> EDTA-NaOH pH 8.0 </td><td> 5mM </td></tr><tr><td> SDS </td><td> % 0.5 (w / v) </td></tr><tr><td> Komple proteaz inhibitörü </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Antikor bağlanma tampon </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 50mm </td></tr><tr><td> EDTA-NaOH pH 8.0 </td><td> 1mm </td></tr><tr><td> NaCl </td><td> 150mm </td></tr><tr><td> Triton X-100 </td><td> % 0,1 (w / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Düşük tuz yıkama tamponu </td></tr><tr><td> NaCl </td><td> 150mm </td></tr><tr><td> SDS </td><td> % 0,1 (w / v) </td></tr><tr><td> Triton X-100 </td><td> % 1 (w / v) </td></tr><tr><td> EDTA-NaOH pH 8.0 </td><td> 2mm </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 20mm </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Yüksek tuz yıkama tamponu </td></tr><tr><td> NaCl </td><td> 500mm </td></tr><tr><td> SDS </td><td> % 0,1 (w / v) </td></tr><tr><td> Triton X-100 </td><td> % 1 (w / v) </td></tr><tr><td> EDTA-NaOH pH 8.0 </td><td> 2mm </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 20mm </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> LiCl yıkama tamponu </td></tr><tr><td> Lityum klorür </td><td> 250mm </td></tr><tr><td> Nonidet-P40 </td><td> % 1 (v / v) </td></tr><tr><td> Sodyum desoxycholate </td><td> % 1 (w / v) </td></tr><tr><td> EDTA-NaOH pH 8.0 </td><td> 1mm </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 20mm </td></tr></tbody></table><table border="1"><tbody><tr><td> Te yıkama tamponu </td></tr><tr><td> EDTA-NaOH pH 8.0 </td><td> 10mM </td></tr><tr><td> Tris-HCl, pH 8.0 </td><td> 1mm </td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinking tampon </td></tr><tr><td> Tris-HCl, pH 6.8 </td><td> 62.5mm </td></tr><tr><td> NaCl </td><td> 200mm </td></tr><tr><td> SDS </td><td> % 2 (w / v) </td></tr><tr><td> DTT </td><td> 10mM </td></tr></tbody></table> 6. Temsilcisi sonuçları: Arabidopsis thaliana PR2 savunma gen ekspresyonu bir β kodlamaAntimikrobiyal aktivite 12, 1,3-Glukanaz benzothiadiazole (BTH), bitki hormonu salisilik asit 3 sentetik bir analog tarafından sağlandı. Şekil 1 PR2 ifade aktivasyonunun gösterir histon lizin kalıntılarının asetilasyon bir artış ile ilişkili PR2 organizatörü H3 ve H4. Nucleosome yoğunluğu 13 gen aktivasyonu sırasında azalır histon 3 değişmeyen bir bölge için bir antikor ile elde edilen sinyalin, histon asetilasyonu değerleri normalize edildi. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Şekil 1"/> Şekil 1 BTH Arabidopsis PR2 organizatörü histon asetilasyonu neden olur. Bitkiler 100μM BTH veya ıslatılabilir toz kontrolü ile tedavi edildi. 72h sonra, yapraklar toplanmış ve kromatin izole edilmiştir. Asetilasyon özel antikorlar (resimde gösterildiği gibi) ile yağış sonrası elde edilen sinyal histon H3 değişmeyen bir etki alanı için bir antikor ile yağış elde sinyaline normalize edildi. Çöktürülmüş kromatin RT-qPCR tarafından belirlendi. Veri BTH tedavi yokluğunda histon değişiklik seviyeleri için standardize edilmiştir.

<ol>
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Çıkar çatışması ilan etti.

Protokolde en kritik adımlar, DNA ve histon, yaprak malzemenin türü ve miktarı, malzemenin taşlama, ve kromatin sonication crosslinking. Bu konularda daha ayrıntılı bir tartışma için, refs bakın. 9 ve 10. Sonikasyon ve crosslinking: 400bp hakkında parçaları sonication sırasında kromatin bozmak için önemlidir. Ise yetersiz sonication uzun kromatin parçaları bırakacaktır kapsayan sonication, DNA ve histon bozarak kromatin yok edecektir. Distal histon modifikasyonları test lokus yerel değişiklikler ayırt edilemez, çünkü bu yöntemin özgüllüğü etkileyebilir. Sonikasyon verimliliği en iyi örneklerinden DNA izole ve uzunluğu makaslanmış DNA agaroz jel elektroforezi ile belirlenmesi, Sonikasyon, de-crosslinking DNA ve histon önce ve sonra 20μL kromatin toplayarak test edilir. Kromatin hazırlık hala parçalanmış DNA görselleştirme etkileyebilir arıtma bu aşamada, büyük miktarda RNA içerdiğinden RNAse elektroforez önce numuneleri eklenmelidir. Makaslanmış Kromatin DNA DNA 400bp etrafında maksimum sinyal yoğunluğu olmayan makaslanmış Kromatin DNA 10kbp daha uzunsa bir karalama formları ise örnekleri, kromatin immunoprecipitation için yararlıdır. Biz rutin olduğu gibi bırakır kromatin crosslinking için% 3 (v / v) formaldehit kullanır, ancak% 1 (v / v) formaldehit, çoğu durumda yeterli olabilir. Bizim ellerde, düşük formaldehit konsantrasyonları sonucu PCR reaksiyonlarında kısa sonication kez ve arka plan sinyalleri için izin verir. Ancak, yeterli miktarda formaldehit genellikle bağımsız deneyler arasındaki PCR sinyali düşük tekrarlanabilirlik. Bu düşük konsantrasyonlarda formaldehid ile yaprak yetersiz nüfuz etmesine bağlı olabilir. Bileşik süre ile polimerize eğilimi sık sık formaldehit hisse senedi alışverişi yapmak emin olun. Formaldehit çözümleri yalnızca yaklaşık bir ay boyunca stabildir ve bu dönemde pek metanol istikrar uzamıştır. Bu deneysel koşullar kurarken farklı formaldehit konsantrasyonları test etmek için tavsiye edilir. Bitki materyali ve taşlama miktarı: Yaprakların birbirine yapışmasını önlemek için tampon büyük bir hacim düşük miktarda yaprak malzeme sızmak için çok önemlidir. Yaprak formaldehit yetersiz infiltrasyonu genellikle sonuçları yapışmasını. Ayrıca, çok fazla bitki materyali miracloth doku gözenekleri bloke eder. Öğütme verimliliği önemli ölçüde mükemmel bir uyum ile bir havanda kullanarak bağlıdır. Biz rutin olarak ek sıvı azot yokluğunda bir sıvı azot soğutmalı havaneli ve harç ile öğütmek için üç kez 50 saniye kadar malzeme ince bir toz için zemin.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Protein-A-Agaroz </td><td> Roche </td><td> 11 134 515 001 </td><td> antikor sınıfı bağlıdır. </td></tr><tr><td> Protein-G-Agaroz </td><td> Roche </td><td> 11 243 233 001 </td><td> antikor sınıfı bağlıdır. </td></tr><tr><td> Anti-hyperacetyl-H4 </td><td> Millipore </td><td> 06-946 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-asetil-H4K5 </td><td> Millipore </td><td> 07-327 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-asetil-H4K8 </td><td> Millipore </td><td> 07-328 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-asetil-H4K12 </td><td> Millipore </td><td> 07-595 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-asetil-H4K16 </td><td> Millipore </td><td> 07-329 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-asetil-H4K18 </td><td> Millipore </td><td> 07-354 </td><td> biz 1μL kullanılan </td></tr><tr><td> Anti-asetil-H3K9 </td><td> Millipore </td><td> 07-352 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-asetil-H3K14 </td><td> Millipore </td><td> 07-353 </td><td> biz 1μL kullanılan </td></tr><tr><td> Anti-trimetil-H3K4 </td><td> Diagenode </td><td> PAB-003-50 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-dimetil-H3K4 </td><td> Millipore </td><td> 07-030 </td><td> biz 5μL kullanılan </td></tr><tr><td> Anti-monometil-H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5-10μL </td></tr><tr><td> Anti-H3 </td><td> Abcam </td><td> ab1791 </td><td> histon yoğunluğu kontrol etmek için 1μL kullanılan </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 </td><td></td></tr><tr><td> Sonotrode MS72 </td><td> Bandelin </td><td></td><td></td></tr><tr><td> Miracloth </td><td> Calbiochem </td><td> 475855 </td><td></td></tr><tr><td> Komple proteaz inhibitörü </td><td> Roche </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

Kromatin yapısı ökaryotlarda gen ekspresyonunun düzenlenmesi için önemlidir. Bu süreçte, histon H3 ve H4 amino-terminal kuyrukları kromatin remodeling, DNA metilasyon ve kovalent değişiklikler temel rolleri 1-2 oynamaktadır. H3 ve H4 histon modifikasyonları, lisin ve arjinin metilasyon, lisin asetilasyonun, serin kalıntıları 1-2 fosforilasyon içerir . Bu modifikasyonlar gen aktivasyonu, baskı ya da uygun sinyalleri (mikrop-ilişkili moleküler desenleri, ışık, hormonlar, vs.) 3-7 algı sonra ifade daha hızlı ve sağlam bir aktivasyon destekler gen astarlanmalıdır bir devlet ya ilişkilidir . Burada, biz, özel seçilmiş bitki genleri kromatin modifikasyonları güvenilir ve hassas tespiti için bir yöntem mevcut. Teknik değişiklik-spesifik antikorların 9,10 ile 8,9 formaldehit, ekstraksiyon ve kromatin, kromatin immunoprecipitation (ChIP) sonication (modifiye) histon ve DNA çapraz bağlanma dayalı, histon-DNA kompleksleri de-crosslinking gen belirli gerçek zamanlı kantitatif PCR. Yaklaşım, C 4 mısır 5,11 fotosentez ve Arabidopsis 3 sistemik bağışıklık ile ilgili özel histon modifikasyonları tespit için yararlı olduğu kanıtlanmıştır .

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

Özgü bitki genleri histon değişiklikleri algılamak için güvenilir ve faydalı bir yaklaşım açıklanmıştır. Bu yaklaşım, kromatin immunoprecipitation (ChIP) ve gerçek zamanlı kantitatif PCR birleştirir. Çeşitli fizyolojik süreçlerde bir rol ile spesifik genlerin histon modifikasyonları tespiti sağlar.

Bitki Leaves Histon değişiklik tespiti

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

Research

JoVE Journal

Biology

24.8K Görüntüleme.  RWTH Aachen University. Özgü bitki genleri histon değişiklikleri algılamak için güvenilir ve faydalı bir yaklaşım açıklanmıştır. Bu yaklaşım, kromatin immunoprecipitation (ChIP) ve gerçek zamanlı kantitatif PCR birleştirir. Çeşitli fizyolojik süreçlerde bir rol ile spesifik genlerin histon modifikasyonları tespiti sağlar.

Video: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

ELISA yöntemi ile yerli bozulmamış nükleozom Post-translasyonel modifikasyonlar Algılama

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Biyoloji

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Ağ Geçidi Uyumlu Bimoleküler Floresan tamamlama (BiFC) kullanarak Bitki Protein Etkileşimleri Tespiti

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Memeli Linker-histon Alttipleriyle İfadesi Analizi

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plant protein kompleksleri, çevirim sonrası değişikliklerin belirlenmesi

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

Tesisinden Akışkan apoplastik çıkarımı için infiltrasyonu-santrifüj Tekniği Kullanarak bırakır<em&gt; Phaseolus vulgaris</em&gt; Bir örnek olarak

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Kristalin Selüloz Birikimin Yüksek Çözünürlüklü Niceleme<em&gt; Arabidopsis</em&gt; Kökler Doku spesifik Hücre Duvarı Değişiklikler Monitör

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Domates Tüylü Roots Üretimi ile yüksek verim CRISPR Vektör İnşaat ve DNA Değişiklikler Karakterizasyonu

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Histon Ekstraksiyon Gönderen Data Analizi: Aşağıdan yukarıya Kütle Spektrometre kullanarak histon post-translasyonel modifikasyonlar Analizi için komple iş akışı

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hibrid-Cut: Dirençli Bir Bitki Doku Örnekleri Bir Geliştirilmiş Kesit Yöntemi

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...