NU-ELISA yöntemi geliştirilmesi, Ulusal Sağlık Enstitüsü, hibe RO1AG023687 tarafından desteklenen oldu.

1. Memeli Hücresi Kültürü NU-ELISA kültürde yetişen herhangi bir memeli hücre tipi yapılabilir. Biz, böylece birkaç farklı antikorları (Abs) deneyleri izin nükleozom aynı yüklü birkaç ELISA plakaları hazırlamak için yeterli miktarda izole edilebilir, böylece hücrelerin büyük gruplar orta hazırlamak için tercih ediyor. Aşağıdaki kültürü ölçekleri yeterli malzeme sağlar: Fare embyronic kök hücreler, hücre bir ya da iki adet 15 cm tabak besleyici hücreler olmadan büyür. Fibroblastlar için 5 ila 10 15 cm tabak, izdiham büyür. 2. Çekirdeklerin izolasyonu Not: olarak belirtilen hariç tüm adımları önceden soğutulmuş tamponlar ile buz üzerinde. <ol><li> Hücreleri Trypsinize, plaka başına 3 ml tripsin ile birleşir ve buz gibi soğuk PBS / butirat 20 ml ekleyin. 1000 rpm&#39;de 5 dakika boyunca santrifüjleyin. </li><li> 10 ml PBS / butirat hücrelerinin yeniden süspanse edin ve 5 dakika boyunca 1000 rpm&#39;de santrifüj. </li><li> Proteaz inhibitörleri (Sigma-Aldrich P-8340) ile 4 ml lizis tamponu yeniden süspanse edin. B tipi havaneli ile buz üzerinde 20 vuruş homojenize Dounce. </li><li> 4 10 dakika 2000g santrifüjleyin ° C (Süpernatant sitoplazma içerir, bu protokol için gerekli değildir, ancak istenirse, diğer kullanımlar için kaydedilebilir). </li><li> 2 ml buz yıkama tamponu C pelet (proteaz inhibitörleri ile) tekrar </li><li> Katman 5 ml% 30 sukroz minder üzerinde yeniden süspanse malzeme, daha sonra 5 dakika sallanan bir kova rotor 2400g santrifüjlenir. Çekirdekler, yastık yoluyla göç, ve enkaz arayüzü kalır. </li><li> Dikkatle çıkarın ve tüm sıvı hacmi 250 ul buz soğuk yıkama tamponu C + proteaz inhibitörleri çekirdekleri tekrar süspansiyon. </li></ol> 3. Yerinde Micrococcal Nükleazlar (MNase) Sindirim tarafından nükleozom izolasyonu Biz kromatin süpernatantı ücretsiz bozulmamış nükleozom sürücü hipotonik bir tedavi takip MNase ile onları beslerken çekirdeklerin içinde in situ sindirilir bir yordamı kullanın. <ol><li> 3 ul 0.1 M CaCl 2 örnek ekleyin. 37 ° C ısıya blok koyun. 37 ° C sıcaklık varsaymak izin verin. </li><li> 2 adet MNase (2 units/10μl MNase tampon içinde çözünmüş Micrococcal Nükleazlar, Sigma-Aldrich), daha sonra 37 inkübe ° C 12 dakika, bir pipetlemeyin ucunu kullanarak sık karıştırma. </li><li> Reaksiyonu durdurmak için 0.5 M Na-EDTA, pH 8.0 ul 6 ekleyin. Buz koyun. </li><li> Süpernatant atmak için 2000g, 4 dakika santrifüjleyin. 300 ul 0.2 mM Na-EDTA pelletini tekrar. 1 saat buz üzerinde zaman zaman nazik pipeting saklayın. (Bu hipotonik koşulları çekirdekleri ücretsiz nükleozom kurtarmak). </li><li> 4 dakika süreyle 3000 g santrifüjleyin 4 ° C Buz üzerinde, ücretsiz nükleozom içeren süpernatant kaydedin. </li><li> 300 ul 0.2 mM Na-EDTA ile tekrar pelletini tekrar. 1 saat buz üzerinde zaman zaman nazik pipeting saklayın. </li><li> 4 4min için 3000g santrifüjleyin ° C Süpernatantı saklayın ve 5. adımdan itibaren ilk süpernatant ile birleşir. -80 ° C&#39;ye çıkan mononucleosome hazırlıkları aliquoted ve saklanabilir </li></ol> Not: kromatin miktarı kabaca 10 ul örnek 260 nm&#39;de absorbans ölçümü ile quantitated 990 ul su eklenir. A 260 = 10 (1 / 100 dilüsyon için ayarladıktan sonra) kromatin 1mg/ml karşılık gelir. Ayrıca kendi DNA içeriği, ağırlıklı olarak uzunluğu 146 bp DNA içermelidir MNase sindirimler kalite ve ölçüde izlemek için, yukarıdaki prosedürü sırasında, küçük örnekler muhafaza edilmesi ve daha sonra analiz. Laddering eksik MNase sindirim göstergesidir. Not: Şekil 1 nükleer izolasyon ve MNase sindirimler adımları diyagramlanabilmiştir özetini içerir. 4. Nucleosome-ELISA (NU ELISA) Biz 96 ELISA microtiter plakalar üzerinde immobilize edilmiştir yerli bozulmamış nuclesosomes içeren kesirler PTMS algılar. Her bir örnek için, 2 kat dilüsyonlarının bir dizi yapmak ve bu üç nüsha olarak kuyu üzerine kaplanmıştır. <ol><li> 4 ° C 50 ul / kuyu kaplama tampon içinde seyreltilmiş nükleozom. Gecede Coat MaxiSorp plakalar Nükleozom Önerilen seri iki yönlü dilüsyonları sadece alt kaplama tampon (0 mg kromatin), 0.1 mg, 0.05 mg, 0.025 mikrogram, 0.0125 mg, 0.00625 mg, mcg 0,00313 0,00156 mg ekleyerek üst satırdan alt satıra hazırlanan satır. (Not: Bu adım için plakası örtüleri ihtiyaç vardır.) </li><li> Sabah, bir tabak yıkama ile toplam 10 dakika oda sıcaklığında (RT) 200 ul / iyi PBS/0.5% Tween-20 ile plakalar 4 kez yıkayın. </li><li> Blok 1 saat RT 100 ul / Tween-20 /% 5% PBS/0.05 BSA. </li><li> Briskly ters plaka sallayarak engelleme tamponu çıkarınlavabonun üzerinde. Kapak ve -20 ° C&#39;de plakaları mağaza, ya da doğrudan bir sonraki adıma gidin. </li><li> 1 ° Abs PBS/0.05% rotator 1 saat oda sıcaklığında inkübe Tween-20 /% 5 BSA, 50 / ml iyi seyreltilmiş 1:1000 bir ses (ya da gerektiği gibi) ekleyin. </li><li> RT 200 ul / iyi PBS/0.5% Tween-20 ve toplam 10 dakika için bir tabak yıkama plakalar 4 kez yıkayın. </li><li> At turp peroksidaz (HRP) konjuge 2 ° Abs PBS/0.05% rotator bir saat için RT Tween-20 /% 5 BSA (ya da gerektiği gibi) 1:5000 sulandırılmış ekleyin. </li><li> 200 ul / PBS/0.5% Tween-20 ile plakalar 4 kez yıkayın. Her yıkama bir tabak yıkayıcı RT ve 10 dakika olmak üzere toplam. </li><li> RT 10 dakika için her bir kuyunun 50 ul TMB substrat ekleyerek plakalar geliştirin. 50 ul / kuyu 4 SO H 2 2N, ekleyerek reaksiyon durdurun ve plakaları 450 nm okuyun. (İpucu: absorbans okuma önce kuyularda herhangi bir hava kabarcığı dağıtmak için bir tabak rotor kullanarak 2 dakika süreyle 1500 rpm&#39;de Santrifüj). </li><li> Nicel ve istatistiksel analizler için tablo dosyaları okuma ihracat. </li></ol> Reaktifler <table border="1"><tbody><tr><td> PBS / butirat </td></tr><tr><td> 135 mM NaCl </td></tr><tr><td> 2.5 mM KCl </td></tr><tr><td> 8 mM Na 2 HPO 4 </td></tr><tr><td> 1.5 mM KH 2 PO 4 </td></tr><tr><td> 10 mM Na-butirat </td></tr></tbody></table><table border="1"><tbody><tr><td> Lizis Tampon </td></tr><tr><td> 250 mM sakkaroz </td></tr><tr><td> 10 mM Tris-HCl, pH 7.4 </td></tr><tr><td> 10 mM Na-butirat </td></tr><tr><td> 4 mM MgCl 2 </td></tr><tr><td> % 0.1 Triton X-100 </td></tr></tbody></table><table border="1"><tbody><tr><td> C yıkama tamponu </td></tr><tr><td> 250 mM sakkaroz </td></tr><tr><td> 10 mM Tris-HCl, pH 7.4 </td></tr><tr><td> 10 mM Na-butirat </td></tr><tr><td> 4 mM MgCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Sakkaroz Yastık </td></tr><tr><td> % 30 (w / v) yıkama tamponu C sakaroz </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal Nükleazlar Tampon </td></tr><tr><td> 5 mM Napo 4 Tampon, pH 7.0 </td></tr><tr><td> 0.025 mM CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Kaplama tampon </td></tr><tr><td> Çözüm A + 80ml 170ml Çözüm B + 250ml dH 2 O </td></tr><tr><td> Çözüm: 0.2 M Na 2 CO 3 </td></tr><tr><td> Çözelti B: 0.2 M NaHCO 3 </td></tr></tbody></table> 5. Temsilcisi Sonuçlar NU-ELISA yöntemi, kromatin mononucleosomes şeklinde hazırlanmış olan yüklenebilir hazırlanan birkaç aynı plakaları bir dizi kullanma. Biz genellikle, her biri farklı anti-PTM özel antikorlar (Abs) probed böylece aynı yüklü levhalar bir dizi hazırlıyoruz. Biz de her zaman değişiklik devlet toplam kromatin yükleme kontrolü dikkate alınmaksızın histon algılar Ab probed bir tabak hazırlayın. Bu kontrol, daha sonra kantitatif farklı örneklerinden hazırlanan nükleozom PTM içerik karşılaştırmak için şarttır. , Biz bu yaklaşımı kullanarak ham absorbans değerleri düzeltmenize, belirli bir kromatin örneklem içinde PTMS düzeylerini ölçmek için: Öncelikle, biz hiçbir kromatin (arka plan genellikle ihmal edilebilir) içeren kontrol kuyulardan elde edilen değerler kullanılarak herhangi bir arka plan sinyali çıkarın. Daha sonra Ab değişiklik devletin bağımsız nükleozom algılar bir test aynı yüklü plaka elde NU-ELISA sonuçlarına PTM özel sinyaller standartlaştırmak. (Bunun için histon H2A veya H2B için spesifik poliklonal Abs kullandık). Daha sonra, kromatin her seyreltme ve sapmaların belirlemek ve ELISA testinin doğrusal kısmını elde edilen verileri kullanabilir. NU-ELISA matematiksel ve istatistiksel analizler ayrıntılı örnekler, daha önce 4 bildirilmiştir <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Şekil 1"/> Şekil 1. NU-ELISA adımları şematik diagam. A. Memeli Hücreleri hasat; B. Çekirdekler Dounce saat hücreleri ayrılıromogenization, C. hücreler santrifüj sonra% 30 w / v sakaroz yastık, D. üst yüklenen bozulur, çekirdeklerin pelet yatırılır, E, F. Kromatin MNase tarafından ağırlıklı olarak mononucleomes in situ sindirilir, G, H ben Çözünür mononucleosomes hipotonik tedavi ve artık nükleer malzeme tekrarlanan santrifüj tarafından ayıklanır. J. Mononucleosomes farklı örnekleri (etiketli samp Bu durumda 1 ve 2) aynı yüklü levhalar seri mikrotiter kuyuları kaplı ve sorgulandı. toplam kromatin yükleme belirlemek için PTM özel Abs ve PTM bağımsız Abs. K. diferansiyel sinyal algılama düzeyleri, onların PTM içeriği farklı iki örnek tasviri, henüz anti-H2B immünoreaktivite tarafından değerlendirilecek benzer genel kromatin içeriğe sahip .

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

NU-ELISA belirli bir hücre tipi içinde nucleosome PTMS küresel durumunu tespit etmek için bir yöntem sağlar. NU-ELISA çalışmaları küresel nucleosome değişiklik durumları birbirinden farklı hücre tipleri 4 karşılaştırmalar farklı olduğunu göstermiştir. Buna ek olarak, hücreler gibi epigenetik düzenleyici ajanlara maruz kaldığında NU-ELISA PTM profilleri değiştirmek trichostatin-A veya fare embyronic kök hücreler 4 farklılaşmış. Bu yöntem aynı zamanda insan ES hücrelerinin 5 kromatin çalışmada başarıyla uygulanmaktadır . NU-ELISA, bugünkü şekliyle, sadece hücresel Epigenom toplamı içinde PTMS kompozit imza algılayabilir dikkat etmek önemlidir. Bu, belirli genetik lokusların genelinde belirli bir PTM dağılımını belirlemek genom kromatin immunoprecipitation, belirgin farklıdır. NU-ELISA prosedürleri ilk adımları memeli çekirdekleri 6,7 mononucleosomes izole etmek için daha önceki yöntemlerden uyarlanmıştır . Bu uyarlanmış yöntemler, memeli hücreleri yüksek kaliteli sağlam mononucleosomes elde etmek için bir çare bir yol sağlar ve ortaya çıkan kesirler kromatin içeriği kapsamlı, ancak büyük bir ek nükleer malzeme içerir. Özel Abs kullanılan olduğundan, olmayan nucleosomal malzeme kirlenmesine neden olan, daha büyük bir saflık gerektiren kitle-spec yaklaşımların aksine, NU-ELISA testinin iyi tolere edilir. Ancak, NU-ELISA batı western ve nucleosomal PTMS tespiti için kitle spec iyi alternatifler sunan, böylece 4 blot daha duyarlı ve kantitatif.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Micrococcal Nucleae </td><td> Sigma </td><td> N3755 </td><td> -20 ° C&#39;de saklanan 2 U/10μl tampon hacimde yapın </td></tr><tr><td> Nunc MaxiSorp microtiter Tabaklar </td><td> Balıkçı </td><td> 12-565-135 </td><td> Yapıştırıcı plakası örtüleri de Fisher üzerinden sipariş edilebilir </td></tr><tr><td> Otomatik Plaka Yıkama </td><td></td><td></td><td></td></tr><tr><td> 1-Adım TMB-ELISA substrat </td><td> Delmek </td><td> 34028 </td><td> Mağaza, 4 ° C Kullanmadan önce RT öncesi sıcak ya da kâğıt üzerindeki teknik özellikler </td></tr><tr><td> Microtiter Levha Reader </td><td> Bio-Rad mikroplaka okuyucu (model: 680) </td><td></td><td> En kolorimetrik plaka okuyucu modelleri uygundur. </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

Ökaryotlar genomu DNA ve proteinler içeren kromatin olarak var. Kromatin temel birimi, iki histon H2A, H2B, H3, H4 1 ile ilişkili DNA 146 baz çifti içeren nucleosome . Histon, N-terminal kuyruklarını lisin ve arjinin zengin ve post-transkripsiyonel asetilasyon, metilasyon ve diğer post-translasyonel modifikasyonlar (PTMS) tarafından modifiye edilmiştir. Nükleozom PTM yapılandırma dolayısıyla doğa 2,3 epigenetik gen regülasyonu bir mod, ilişkili DNA transkripsiyonel aktivitesini etkileyebilir. Biz nucleosome ELISA (NU-ELISA) hücrelerden çıkarılan nükleozom küresel PTM imzaları belirlemek için kantitatif olarak adlandırılan bir yöntem geliştirdi. NU-ELISA blot batı daha duyarlı ve kantitatif ve özel hücre tipleri epiproteomic durumunu sorgulamak için yararlıdır. Bu video dergi makalesi, NU-ELISA analizi gerçekleştirmek için ayrıntılı yordamlar gösterir.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

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

Nucleosome ELISA (NU-ELISA), post-translasyonel modifikasyonlar, doğal, bozulmamış nükleozom hazırlıkları küresel kalıplarını tespit etmek için hassas ve kantitatif bir yöntemdir. Bu değişiklikler özel histon amino asit artıkları metilasyonla, asetilasyon, fosforilasyon, ve dolayısıyla NU-ELISA belirli hücre tiplerinin genel kromatin modifikasyon devletlerin küresel bir proteomik tahlil sağlar.

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 ...

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JoVE Journal

Biology

17.2K Görüntüleme.  Stanford University. Nucleosome ELISA (NU-ELISA), post-translasyonel modifikasyonlar, doğal, bozulmamış nükleozom hazırlıkları küresel kalıplarını tespit etmek için hassas ve kantitatif bir yöntemdir. Bu değişiklikler özel histon amino asit artıkları metilasyonla, asetilasyon, fosforilasyon, ve dolayısıyla NU-ELISA belirli hücre tiplerinin genel kromatin modifikasyon devletlerin küresel bir proteomik tahlil sağlar.

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

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Zymography Fonksiyonel Matrix Metalloproteinazlar Tespiti

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Biyoloji

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Polarize Epitel Hücreleri içine Plazmidler, Mikroenjeksiyon Küçük GTPases Fonksiyon Analizi

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

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 ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Bozulmamış Arterlerin endotel katmanı Nitrik Oksit ve Süperoksit En Yüz Algılama

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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 ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Bozulmamış Bitkiler Ağartma veya Ekstraktlarının Isıl İşlem tarafından Tütün Ana Hücre Proteini Temizleme Yöntemlerinin Karşılaştırılması

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...