Servane Contal ve Boris Untereiner Biz kendi teknik katkılarından dolayı teşekkür ederim. Biz FNR &quot;FUTOX&quot; projesi destek kabul etmiş sayılırsınız.

Protokol, tamamlanması için 16 gün gereklidir. Zaman diliminde Şekil 1&#39;de ayrıntılı olarak verilmiştir. Ayrıntılar tampon hazırlık <ul><li> Lizis tamponu (örnek başına 3ml) hazırlanması. </li><li> Yıkama tamponu (örnek başına 50ml) hazırlanması. Bu tampon önceden soğutulmuş ve saklanan derin dondurucuda -20 ° C&#39;de daha fazla analiz kadar ve tüm prosedürü sırasında buz üzerinde tutulmalıdır olmalıdır. </li><li> Yağış tamponu (örnek başına 20ml) hazırlanması. Bu tampon, bütün prosedür sırasında gerekli ve buz üzerinde tutulmalıdır kadar derin dondurucuda -20 ° C&#39;de önceden soğutulmuş ve saklanan olmalıdır. </li><li> 4 iş tercihen soğuk odasına yürütülen olmalıdır ° C </li></ul> Mantar türlerinin Proteomik analizleri için kullanılan üç tür morfolojik özellikleri ve Çeviri uzama faktörü Alpha-1 analizi temelinde Fusarium graminearum sınıflandırıldı (O&#39;Donnell ve ark, 1998) Bu suşlar CRP-Gabriel Lippmann koleksiyonuna aitti ve korunur ve muhafaza edildi - 80 ° C 15% gliserol. Miseller, hazırlanması Mantarlar V8 25 ° C ışık ve karanlık bir dönem değişen her 12 saat, 4 gün boyunca yetiştirildi. Kültürler, steril bir bıçak ve bu kültürlerin adet 100 ml toksin tetikleyen medya içeren 250 ml Erlenmeyer şişeler aşılamak için kullanılmıştır kullanarak parçalandı. Miçel 10 gün sonra hasat edildi ve steril bir filtre ile kültür filtre ederek ortamdan ayrıldı. Miçel orta artıkları ve diğer bileşikler kaldırmak için steril su ile 3 kez yıkandı. Daha sonra hücrelerin sıvı azot ekleyerek hemen dondu ve -80 örnekleri tuttu ° C Protein ekstraksiyon Genel açıklama Protein örnekleri rakam 3A ve 3B açıklanan yöntem kullanılarak çıkarıldı. Fusarium örnekleri, sıvı azot zemin ve, toz, 10 ml teflon tüpler toplandı. Örnekler daha sonra lizis tamponu ile 30 dakika inkübe 10 dakika kaynatılır ve oda sıcaklığında (15 dakika için 12000g) 2 kez santrifüj edildi. Süpernatantlar -20 ° C&#39;de bir gece yağış tamponu ile toplanır ve inkübe edildi. 4 ° C 30000g santrifüj sonrasında 45 dakika, çöktürülmüş proteinlerin pelet DTT içeren soğuk aseton ile 3 kez yıkandı. Son olarak, pelet hava kuru ve proteinlerin pH 8 &#39;e ayarlayarak, etiketleme tampon resolubilized edildi. 30 ml&#39;lik bir alt-örneklem total protein ölçümü için çıkarıldı ve kalan süpernatant protein elektroforezi kadar -20 ° C&#39;de saklanır. <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="Şekil 1" /> Şekil 1 prosedürü Timeline. <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="Şekil 2" /> Şekil 2 birden fazla operatör örnek işleme Programı. <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="Şekil 3a" border="0" /> Şekil 3a. <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="Şekil 3b" border="0" /> Şekil 3b. Lütfen <a href="/files/ftp_upload/1690/1690fig3a.jpg">buraya tıklayın</a> daha büyük bir rakam 3a sürümünü ya da görmek için <a href="/files/ftp_upload/1690/1690fig3b.jpg">buraya</a> , daha büyük bir rakam 3b versiyonu için . Şekil 3a-b protein ekstraksiyon prosedürü Akış şemaları (A: ilk gün; B: ikinci gününde). <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="Şekil 4" border="0" /> Şekil 4 protein özleri 1D resim. Proteinler lav Purple ile boyandı jel sonuna kadar mavi tam göç kadar çalıştırmak. Lütfen Şekil 4 büyük halini görmek için <a href="/files/ftp_upload/1690/1690fig4.jpg">buraya tıklayın</a> .

<ol>
	<li>Bridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Extraction+from+Fungi.">Protein Extraction from Fungi.</a> Protein Purification Protocols. , 39-48 (1996).</li><li>Jiao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+carbon+sources+on+trichothecene+production+and+Tri+gene+expression+by+Fusarium+graminearum+in+liquid+culture.">Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture.</a> FEMS Microbiol Lett. 285, 212-219 (2008).</li><li>Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+filamentous+fungi.">Proteomics of filamentous fungi.</a> Trends in Biotechnology. 25, 395-400 (2007).</li><li>Milles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+proteomic+approach+to+monitor+protein+synthesis+in+mycotoxin+producing+moulds.">Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.</a> Mycotoxin Res. 23, 161-165 (2007).</li><li>Morrison, N., Field, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concept+of+sample+in+OMICS+technology.">Concept of sample in OMICS technology.</a> OMICS. 10, 127-137 (2006).</li><li>O'Donnell, K., Kistler, H. C., Cigelnik, E., Ploetz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+evolutionary+origins+of+the+fungus+causing+Panama+disease+of+banana:+Concordant+evidence+from+nuclear+and+mitochondrial+gene+genealogies.">Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies.</a> PNAS. 95, 2044-2049 (1998).</li><li>Paper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+of+extracellular+proteins+in+vitro+and+in+planta+from+the+pathogenic+fungus+Fusarium+graminearum.">Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.</a> Proteomics. 7, 3171-3183 (2007).</li><li>Pasquali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+in+science.+Protocol+videos:+the+implications+for+research+and+society.">Video in science. Protocol videos: the implications for research and society.</a> EMBO Rep. 8, 712-716 (2007).</li><li>Taylor, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+information+about+a+proteomics+experiment+(MIAPE).">The minimum information about a proteomics experiment (MIAPE).</a> Nat Biotechnol. 25, 887-893 (2007).</li><li>Taylor, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analyses+of+Fusarium+graminearum+grown+under+mycotoxin-inducing+conditions.">Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions.</a> Proteomics. 8, 2256-2265 (2008).</li></ol>

Mantar biyoloji etki alanı içinde proteomik yaklaşımlar son zamanlarda ilgi, bu tekniği kullanarak (as Kim ve ark, 2007 gözden) yayınların sayısının artmasına yol açtı . Protein ekstraksiyon yöntemleri çıkarma prosedürlerinin kombinasyonu güveniyor; genellikle pek metodolojik bölümlerde açıklanan ve potansiyel sorun giderme yöntemleri ile ilgili uzmanlık gerektirir. , Örnek ve veri işleme için standartları belirleyen diğer &quot;omics&quot; yaklaşımlar, güvenilir bilimsel bilgi üretmek için esastır. Ayrıca manipülasyon örnekleri ve prosedürleri farklı &quot;omics&quot; deneyler sonucu yorumlama arasında paylaşılan izin vermek için yeterince tarif edilmelidir. Bu tamamen genomik, proteomik, metabolomik, çapraz veri madenciliği potansiyellik istismar için gerekli olacaktır ... (Morrison ve ark, 2006). Proteomik bir denemeyle ilgili asgari bilgileri bir denemenin bütün yönleriyle (Jel elektroforezi, Kütle Spektroskopisi, Moleküler Etkileşimler, Protein Değişiklikler, Proteomik Bilişim, Örnek İşleme) mücadele amacıyla hazırlanmıştır ve çalışma grupları kurulmuştur. Şu anda herhangi bir tanımlı bilgilerle örnek işleme prosedürleri mevcuttur. (Taylor ve ark, 2007) MIAPE kurallar çerçevesinde standardizasyon artırabilir prosedürleri uygulamak için, proteomik çalışmalar son kalitesini belirleyen protein çıkarma prosedürlerinin önemi göz önüne alındığında, biz örnek ayrıntılı bir video protokol önerdi işleme. Deneylerin video tanımı &quot;omics&quot; deneyler (Pasquali 2007) daha iyi tekrarlanabilirlik neden olabilir numune işleme hakkında bilgi sayısını artırmak için önemli ölçüde katkıda bulunabilir. Gerçekten de, laboratuvarlar boyunca tekrarlanabilirliği proteomik sonuçları (http://www.fixingproteomics.org) geçerliliğini garanti altına almak için temel bir gerekliliktir. Farklı instrumentations ve farklı operatörler örnekleri manipüle: çapraz laboratuvar tekrarlanabilirliği iki faktör tarafından etkilenmektedir. Açıklanan protokol, biz gerçekleştirmek için teklif biyolojik veriler (yani sonuçların teknik değişimi dikkate alınır) güvenilirliğini artırmak için birden fazla operatörleri (Şekil 2 ve video) içeren çoğaltır. Protein ekstraksiyonu için açıklanan prosedür SDS ve ısıtma dayanmaktadır. Bu proteinlerin iyi bir saflık ve miktar (Bridge 1996), daha önce garanti gösterildi. SDS kaynatılarak ile birleştiğinde hücre duvarları, hidrofobik proteinler çözünür ve proteinlerin iptal yağış oligomerler oluşumunu önler. Kaynama proteazların inaktivasyonu da sağlar. Aynı etki, EDTA, PMSF ve tam mini proteaz inhibitörü tarafından da üretilir. DTT ve protein çözünmüş kolaylaştırılması proteinler arasındaki disülfit köprüleri kaldırır. IEF önce SDS kaldırmak için, proteinler, aseton / TCA / DTT çöktürülür. Ayrıca, bu bileşiklerin mevcut olduğunda, yağlar, nükleik asitler, tuzlar veya / ve fenolik bileşikler gibi bazı yabancı maddelerden kaldırarak aseton / TCA / DTT yağış sağlar. SDS gibi, bu molekülleri IEF sırasında iyi bir göçü önlemek. Bu yağış sonrasında, IEF ile müdahale gibi TCA kaldırmak için aseton / DTT, çöktürülmüş proteinleri yıkamak için gereklidir. İyi bir numune hazırlama, iyi sonuçlar için bir anahtardır. Bunun için, çevre proteinlerin toz ücretsiz eldiven ile çalışan ve iyi laboratuvar uygulamalarına saygı kirlenmesini önlemek için de gereklidir. Teknik açıdan, açıklanan protokol dahilinde, iki aşamada proteinlerin yeterli miktarda ve saflık elde etmek için en kritik adımlar. İlk olarak, öğütme hücrenin tam imha proteinler serbest bırakmak için temel aşaması ve ikinci, toplam proteinlerin saflaştırılması, lipid, DNA ve proteinlerin göç ile parazite neden olabilecek diğer kirleticiler çoğunluğu kaldırılması kapsayan bir faz.

Medya ve çözümler PDA: 39 g Patates Dekstroz Agar (Difco), 1 L distile su. V8 Agar: 200 ml V8 (Campbell, ABD), 2g CaCO3 (Sigma), 16g Agar (Difco), 800 ml distile su Toksin indükleyici medya: 1g K 2 HPO 4, KCl 0.5g, 0.5g MgSO 4 7H 2 O, 10 mg FeEDTA, 2g L glutamik asit, distile su 1L 10g sakaroz. Lizis Tampon: <table border="1"><tr><td></td><td> 100 mL </td><td> Final konsantrasyonu </td><td></td></tr><tr><td> Tris-HCL pH8 </td><td> 5ml </td><td> 50mm </td><td> 1M </td></tr><tr><td> SDS </td><td> 2G veya 10ml </td><td> % 2 </td><td> % 20 </td></tr><tr><td> DTT </td><td> 500ml </td><td> 10mM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20ml </td><td> 0.1mm </td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200ml </td><td></td><td></td></tr><tr><td> Komple mini proteaz inhibitörü </td><td> 1 tablet </td><td></td><td></td></tr><tr><td> Su </td><td> QSP </td><td></td><td></td></tr></table> Yağış Tampon: <table border="1"><tr><td></td><td> 100 mL </td><td> Final konsantrasyonu </td></tr><tr><td> TCA </td><td> 20ml </td><td> % 20 </td></tr><tr><td> DTT </td><td> 0.1ml </td><td> % 0.1 </td></tr><tr><td> Aseton </td><td> QSP </td><td></td></tr></table> Tampon Yıkama: <table border="1"><tr><td></td><td> 100 mL </td><td> Final konsantrasyonu </td></tr><tr><td> DTT </td><td> 0.1ml </td><td> % 0.1 </td></tr><tr><td> Aseton </td><td> 100 mL </td><td></td></tr></table> Etiketleme Tampon (-18 ° C küçük alikotları mağaza): <table border="1"><tr><td></td><td> 1mL </td><td> Final konsantrasyonu </td></tr><tr><td> Üre </td><td> 140 mg </td><td> 7M </td></tr><tr><td> Tiyoüre </td><td> 50 mg </td><td> 2M </td></tr><tr><td> Chaps </td><td> 40 mg </td><td> % 4 </td></tr><tr><td> Tris </td><td> 30 mcL </td><td> 30 mm </td></tr><tr><td> Su </td><td> 1 ml </td><td></td></tr></table>

toxin induction and protein extraction from fusarium spp. cultures for proteomic studies

Fusaria farklı toksinler üretebiliriz ipliksi mantarlar. Fusarium mikotoksinler deoksinivalenol, nivalenol, T2, zearelenone, fusaric asit, moniliformin, vb gibi .. insan hem de hayvan sağlığı üzerindeki olumsuz etkileri ve bazı patojenite faktörleri olarak kabul edilmektedir. Proteomik çalışmalar Fusaria potansiyel patojenik faktörler (Kağıt ve diğerleri, 2007, Houterman ve ark, 2007) tanımlamak için toksin üretimi mekanizmaları (Taylor ve ark, 2008) yanı sıra çözmekte etkili olduğunu gösterdi. Bu nedenle, protein ekspresyonu deneyler, zorlama ve laboratuarlar arasındaki gerçek farklar bulundu güvenmek için proteomik çalışmalar arasında bir karşılaştırma için güvenilir yöntemler kurmak için temel haline gelir. Açıklanacaktır işlemi iki şekilde proteomik prosedürlerin standartlaştırılması artan bir düzeyde katkıda bulunmalıdır. Filme protokol tam olarak tarif edilebilir ayrıntıları seviyesini artırmak için kullanılır. Ayrıca, biyolojik bir süreç standart prosedürlerin mevcut ekstraksiyon prosedürü teknik tekrarlanabilirlik içinde insan faktörü de dikkate alınarak, daha yüksek bir veri sağlamlığı garanti çoğaltır. Kültürler ve protein ekstraksiyonu (Şekil 1) için iki gün için ondört gün açıklanan protokol, onun tamamlanması için 16 gün gerektirir. Kısaca, Fusarium suşları 4 gün boyunca katı ortam üzerinde yetişen, daha sonra el ile parçalanmış ve 10 gün boyunca, modifiye edilmiş bir toksin indükleyici medya (Jiao ve ark, 2008). aktarılır. Miselyum Miracloth bir katmanı sayesinde filtrasyon ile toplanır. Taşlama soğuk bir odasında yapılır. Farklı operatörler çıkarma yapılan teknik farklılıkları (Şekil 2) nedeniyle önyargı dikkate almak için (n = 3) çoğaltır. Ekstraksiyon Taylor ve ark açıklandığı gibi bir SDS / DTT tampon dayalı. (2008) hafif değişiklikler. Toplam protein izolasyonu Aseton / TCA / DTT tampon gecelik ve DTT / Aseton yıkama (Şekil 3a, 3b) kullanarak proteinlerin bir yağış süreci gerektiriyordu. Proteinler sonunda, protein etiketleme tampon resolubilized ve ölçüldü. Ekstraksiyon Sonuçları 2D jeller (IEF / SDS-PAGE) geçmeden önce, bir 1D jel (Şekil 4, SDS-PAGE) görüntülendi. Aynı prosedür, büyüyen diğer medya ve diğer ipliksi mantarlar (Miles ve ark, 2007) proteomik analizler için uygulanabilir .

Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein ekstraksiyonu mantar türlerinde proteomik analizleri için en az bir proteomik deney (MIAPE) kuralları hakkında bilgi göre, başarılı olmak için standardizasyon yüksek düzeyde gerektirir. Biz toksin indüksiyon ve protein izolasyonu sırasında deneysel önyargı en aza indirmek için bir prosedür içeren bir video protokol mevcut<em&gt; Fusarium spp.</em

Toksin İndüksiyon ve Protein Ekstraksiyonu Fusarium Spp.</emProteomik Araştırmalar> Kültürler

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

toxin-induction-protein-extraction-from-fusarium-spp-cultures-for

Nanyang Technological University

Research

JoVE Journal

Biology

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

16.9K Görüntüleme.  Centre de Recherche Public-Gabriel Lippmann. Protein ekstraksiyonu mantar türlerinde proteomik analizleri için en az bir proteomik deney (MIAPE) kuralları hakkında bilgi göre, başarılı olmak için standardizasyon yüksek düzeyde gerektirir. Biz toksin indüksiyon ve protein izolasyonu sırasında deneysel önyargı en aza indirmek için bir prosedür içeren bir video protokol mevcut Fusarium spp.

Video: Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

RNA veya Protein Ekstraksiyonu Özgül Beyin Bölgesi&#39;nden Rat Beyin Mikro-disseksiyon

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Biyoloji

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Proteinlerin Elektrogöçer Ayırma

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

Röportaj: Protein Katlama ve Nörodejeneratif Hastalıklar Çalışmaları

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Nöron ve Glia Geliştirme Çalışmaları Rat Beyin Agrega Kültürler hazırlanması

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Otomatik Proteomik İşleme Dijital Mikroakiskan

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Genetik ve epigenetik Analizleri Parafin Gömülü Malzeme DNA Ekstraksiyon

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Cinsel Gelişim ve askopor Deşarj Fusarium graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Mukayeseli Proteomik Analizi Metabolik Etiketleme ve Membran Fraksiyonasyon<em&gt; Arabidopsis thaliana</em&gt; Süspansiyon Hücre Kültürleri

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Toksin İndüksiyon ve Protein Ekstraksiyonu Fusarium Spp. Kültürler