Bu çalışma ANR-10-IDEX-0001-02, LabEx Cell'n'Scale ANR-11-LBX-0038 ve Institut de convergence Q-life ANR-17-CONV-0005 tarafından desteklenmiştir. CJ, Institut Curie tarafından desteklenmektedir, Fransız Ulusal Araştırma Ajansı (ANR) ANR-12-BSV2-0007 ve ANR-17-CE13-0021 ödüllerini, Institut National du Cancer (INCA) hibe 2014-PL BIO-11-ICR-1 ve Fondation pour la Recherche Medicale (FRM) hibe DEQ20170336756. MMM, Fondation Vaincre Alzheimer hibeSI FR-16055p ve Fransa Alzheimer hibesi AAP SM 2019 n°2023 tarafından desteklenmektedir. JAS, Marie Skłodowska-Curie hibe anlaşması No 675737 ve FRM hibe FDT201904008210 kapsamında Avrupa Birliği'nin Horizon 2020 araştırma ve yenilik programı tarafından desteklenmiştir. SB, FRM hibesi FDT201805005465 tarafından desteklenmiştir.
Janke laboratuvarının tüm üyelerine, özellikle J. Souphron'a, G. Lakisic'e (Institut MICALIS, AgroParisTech) ve A. Gautreau'ya (Ecole Polytechnique) protokolün kurulması sırasında yardımları için teşekkür ederiz. Institut Curie hayvan tesisine fare yetiştiriciliği ve bakımı konusunda yardımları için teşekkür ederiz.
J. Frankel ve M. Nelson tarafından geliştirilen 12G10 antikor, NICHD himayesinde geliştirilen ve Iowa Üniversitesi tarafından sürdürülen Gelişimsel Çalışmalar Hybridoma Bankası'ndan elde edildi.

<ol>
	<li>Verhey, K. J., Gaertig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tubulin+code.">The tubulin code.</a> Cell Cycle. 6 (17), 2152-2160 (2007).</li><li>Janke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tubulin+code:+Molecular+components,+readout+mechanisms,+and+functions.">The tubulin code: Molecular components, readout mechanisms, and functions.</a> Journal of Cell Biology. 206 (4), 461-472 (2014).</li><li>Janke, C., Magiera, M. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylated+tubulin+is+essential+for+touch+sensation+in+mice.">Acetylated tubulin is essential for touch sensation in mice.</a> Elife. 5, (2016).</li></ol>

Yazarların açıklayacak bir şeyi yok.

Burada açıklanan yöntem, hücre çizgilerinden ve tek fare beyinlerinden orta-büyük miktarlarda hızlı bir şekilde yüksek kaliteli, montaj yetkin tubulin üretmek için bir platform sağlar. Uzun yıllardır sahada kullanılan sığır beyinlerinden tübulin saflaştırmanın altın standart protokolüne dayanmaktadır16,17. Yaklaşımın özel bir avantajı, kurulduktan sonra çok az uygulamalı zaman gerektirirken büyük miktarlarda hücre üreten HeLa...

Mikrotübüller birçok hücresel işlemde kritik roller oynar. Hücrelere şekillerini verirler, kromozom ayrımı için meiotik ve mitotik iğler oluştururlar ve hücre içi taşıma için izler görevi ederler. Bu farklı işlevleri yerine getirmek için mikrotübüller kendilerini farklı şekillerde düzenlerler. Alandaki merak uyandırıcı sorulardan biri, yapısal ve evrimsel olarak korunmuş mikrotübüllerin bu organizasyon ve işlev bolluğuna uyum sağlamasını sağlayan moleküler mekanizmaları anlamaktır. Potansiyel mekanizmalardan biri, 'tübulin kodu'1,2,3olarak bilinen kavram tarafından tanımlanan mikrotübüllerin çeşitlendirilmesidir. Tubulin kodu iki ana bileşen içerir: α ve β-tubulin gen ürünlerinin (tubulin izotipleri) mikrotübüllere ve tübulin posttranslational modifikasyonlara (PTM' ler) diferansiyel olarak dahil edildi.
1970'lerden bu yana, in vitro rekonstrüs deneyleri, gelişen ışık mikroskopi teknikleri ile birlikte, mikrotübüllerin özellikleri hakkında önemli keşiflerin önünü açmıştır: dinamik dengesizlik4 ve koşu bandı5ve diğer mekanizmaları ve işlevleri 6,7,8,9,10,11,12,13,14,15. Şimdiye kadar yapılan hemen hemen tüm in vitro deneyler, tekrarlanan polimerizasyon ve depolimerizasyon döngüleri kullanılarak beyin dokusundan saflaştırılmış tübulin bazlıdır16,17. Beyin dokusundan arınma, yüksek kaliteli tübulin elde etmenin avantajını büyük miktarlarda (genellikle gram miktarlar) sağlasa da, beyin dokusundan saflaştırılmış tübulin farklı tubulin izotiplerinin bir karışımı olduğu ve birçok tübulin PTM'si ile zenginleştirilmiş olması nedeniyle önemli bir dezavantaj heterojenliktir. Bu heterojenlik, mikrotübül özelliklerinin ve işlevlerinin kontrolünde belirli bir tübulin PTM'nin veya izotipin rolünü tanımlamayı imkansız kılar. Bu nedenle, tübulin kodunun moleküler mekanizmalarını ele almak için kontrollü tübulin PTM'leri ve homojen izotip bileşimi ile montaja yetkin tubulin üretmek esastır.
Son zamanlarda, maya Stu2p'in mikrotübül bağlayıcı TOG (tümör aşırı ekspresyonlu gen) etki alanını kullanarak tubulin'i benzeşim kromatografisi ile arındırmak için bir yaklaşım geliştirilmiştir18. Bu yöntemde, hücrelerin veya dokunun kabalyatlarındaki tübulin, matris immobilize TOG etki alanına bağlandığı bir sütundan geçirilir, bu da belirli bir, hatta çok küçük bir örneğin tüm tübulin havuzunun analizini sağlar. Rekombinant tübulin saflaştırmak için uzun zamandır beklenen bir yaklaşım da son yıllarda tanımlanmıştır. α ve β-tubulin genleri içeren iki cistronic vektörün böcek hücrelerinde ifade edildiği baculovirüs sistemine dayanmaktadır19. Bununla birlikte, bu yöntem çok hantal ve zaman alıcıdır ve bu nedenle çoğunlukla tubulin mutasyonları20 ve tubulin izotipleri21 , 22,23in vitro etkisini incelemek için kullanılır.
Mevcut protokolde, hücre çizgilerinden veya fare beyin dokusundan farklı modifikasyon seviyelerine sahip tübulin oluşturmak için köklü ve yaygın olarak kullanılan polimerizasyon-depolimerizasyon yaklaşımını kullanan bir yöntemi bir plan olarak tanımlıyoruz24. Bu prosedürde, tübulin çözünür (4 °C'de tübulin dimer) ve polimerize form (guanosin 5'-trifosfat [GTP] varlığında 30 °C'de mikrotübül) arasında döngüye girer. Her form ardışık santrifüjleme adımlarıyla ayrılır: tübulin dimerler soğuk (4 °C) bir dönüşten sonra süpernatanta kalırken, mikrotübüller 30 ° C'de peletlenecektir. Ayrıca, mikrotübül ilişkili proteinlerin mikrotübüllerden ve böylece son olarak saflaştırılmış tübulinden uzaklaştırılmasını sağlayan yüksek piperazin-N,N′-bis(2-etanesülfonic asit) (PIPES) konsantrasyonda bir polimerizasyon adımı gerçekleştirilir. Süspansiyon veya yapışık kültürler olarak yetiştirilen HeLa S3 hücrelerinden arındırılmış Tubulin, herhangi bir tübulin PTM'sinden neredeyse ücretsizdir ve son in vitro reconstitution deneylerindekullanılmıştır 25,26,27,28. Tubulin izotiplerinde ve PTM'lerde değişikliklerle çok sayıda fare modeli için kullanılabilecek tubulin'i tek fare beyinlerinden arındırma yöntemini daha da uyarladık.
Protokolde, ilk olarak kaynak materyalin (hücre kütlesi veya beyin dokusu), lizizinin(Şekil 1A)neslini ve ardından tübulini arındırmak için tübulin polimerizasyonu ve depolimerizasyonunun ardışık adımlarını açıklıyoruz (Şekil 1B). Saflaştırılmış tübulinin saflığını (Şekil 2A,B) ve miktarını (Şekil 3A,B) değerlendirmek için süreci daha ayrıntılı olarak açıklıyoruz. Yöntem, tübulin saflaştırmadan önce hücrelerde bir modifiye enzimi aşırı ifade ederek seçilen bir PTM ile zenginleştirilmiş tübulin üretmek için uyarlanabilir (Şekil 4B). Alternatif olarak, saflaştırma işlemi sırasında tübulin modifiye edici enzimler tubulin eklenebilir. Son olarak, spesifik izotiplerden veya PTM'lerden yoksun tübulini, ilgili tübulin değiştirici enzimlerde eksik olan farelerin beyinlerinden arındırabiliriz (Şekil 4B)29.
Burada tarif ettiğimiz yöntemin iki temel avantajı vardır: (i) nispeten kısa sürede yeterince büyük miktarlarda tübulin üretimine izin verir ve (ii) spesifik tubulin izotip bileşimi veya PTM'leri ile yüksek kaliteli, saf tubulin üretir. Bu makalenin ilişkili videosunda, bu prosedürde yer alan bazı kritik adımları vurguluyoruz.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#160;M MgCl2&#160;</td>
			<td>Sigma</td>
			<td>#M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>1-L cell culture vessels</td>
			<td>Techne F7610&#160;</td>
			<td></td>
			<td>Used for spinner cultures. Never stir the empty spinner bottles. When spinner bottles are in the cell culture incubator, always keep the lateral valves of spinner bottles slightly open to facilitate the equilibration of media with incubator&#8217;s atmosphere. After use, fill the spinner bottles immediately with tap water to avoid drying of remaining cells on the bottle walls. Wash the bottles with deionised water, add app 200 ml of deionised water and autoclave. Under a sterile cell culture hood remove the water and allow the bottles to dry completely, still under the hood, for several hours. Never use detergents for cleaning the spinner bottles because any trace amounts of the detergent can be deleterious to the cells.</td>
		</tr>
		<tr>
			<td>1.5- and 2-ml tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14-ml round-bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm-diameter sterile culture dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#M3148</td>
			<td>2-mercaptoethanol is toxic and should be used under the hood.</td>
		</tr>
		<tr>
			<td>4-(2-aminoethyl)-benzenesulfonyl fluoride&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A8456</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>5-, 10- 20-ml syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-ml, 10-ml, 25-ml sterile pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>#A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin antibody, 12G10&#160;</td>
			<td>Developed by J. Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa</td>
			<td></td>
			<td>dilution: 1/500</td>
		</tr>
		<tr>
			<td>Anti-glutamylated tubulin antibody, GT335&#160;</td>
			<td>AdipoGen&#160;</td>
			<td>#AG-20B-0020</td>
			<td>dilution: 1/20,000</td>
		</tr>
		<tr>
			<td>Aprotinin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance (0.1 &#8211; 10 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman 1-l polypropylene bottles&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Beckman Avanti J-26 XP centrifuge</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Biological stirrer&#160;</td>
			<td>Techne MCS-104L&#160;</td>
			<td></td>
			<td>Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells</td>
		</tr>
		<tr>
			<td>Bis N,N&#8217;-Methylene-Bis-Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Blender IKA Ultra-Turrax&#174;&#160;</td>
			<td></td>
			<td></td>
			<td>For lysing brain tissue, use 5-mm probe, with the machine set at power 6 or 7. Blend the brain tissue 2-3 times for 15 s on ice.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue&#160;</td>
			<td>Sigma&#160;</td>
			<td>#1.08122</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxypeptidase A (CPA)</td>
			<td>Sigma</td>
			<td>#C9268</td>
			<td>Concentration: 1.7&#160;U/&#181;l</td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator set at 37&#176;C, 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>&#160;Sigma&#160;</td>
			<td>#D8418</td>
			<td>DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection.</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#41965062</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT, DL-Dithiothreitol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Euromedex</td>
			<td>#EU0007-C</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma&#160;</td>
			<td>#E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>#E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell press&#160;</td>
			<td>Thermo electron corporation</td>
			<td>&#160;#FA-078A</td>
			<td>with a #FA-032 cell; for lysing big amounts of cells. Set at medium ratio, and the gauge pressure of 1,000 psi (corresponds to 3,000 psi inside the disruption chamber).</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>&#160;VWR Chemicals&#160;</td>
			<td>#24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>#G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP&#160;</td>
			<td>Sigma&#160;</td>
			<td>#G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block&#160;</td>
			<td>Stuart&#160;</td>
			<td>#SBH130D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells&#160;</td>
			<td></td>
			<td>ATCC&#174; CCL-2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela S3 cells&#160;</td>
			<td>ATCC</td>
			<td>ATCC&#174; CCL-2.2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl )</td>
			<td>VWR</td>
			<td>#20252.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td></td>
			<td></td>
			<td>With fluorescence if cell transfection is to be verified</td>
		</tr>
		<tr>
			<td>Isopropanol&#160;</td>
			<td>VWR&#160;</td>
			<td>#20842.298</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus&#160;</td>
			<td>#101</td>
			<td></td>
		</tr>
		<tr>
			<td>JLA-8.1000 rotor&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>KOH&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P1767</td>
			<td>KOH is corrosive and causes burns; use eye and skin protection.</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#25030123</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory centrifuge for 50-ml tubes</td>
			<td>Sigma</td>
			<td>4-16 K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin&#160;</td>
			<td>Sigma</td>
			<td>#L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropestles</td>
			<td>Eppendorf</td>
			<td>#0030 120.973</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse brain tissue&#160;</td>
			<td></td>
			<td></td>
			<td>Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines.</td>
		</tr>
		<tr>
			<td>Needles 18G X 1 &#189;&#8221; (1.2 X 38 mm</td>
			<td>Terumo</td>
			<td>#18G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 20G X 1 &#189;&#8221; (0.9 X 38 mm</td>
			<td>Terumo</td>
			<td>#20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 21G X 4 &#190;&#8221; (0.8 X 120 mm</td>
			<td>B.Braun</td>
			<td>#466 5643</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma&#160;</td>
			<td>#P7626</td>
			<td>PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions.</td>
		</tr>
		<tr>
			<td>PIPES&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette-boy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotors</td>
			<td>Beckman 70.1 Ti; TLA-100.3; and TLA 55</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis equipment&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>VWR&#160;</td>
			<td>#442444H</td>
			<td>For preparing Laemmeli buffer&#160;</td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>Sigma&#160;</td>
			<td>#L5750</td>
			<td>For preparing &#39;TUB&#39; SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>Sonicator&#160;</td>
			<td>Branson</td>
			<td>#101-148-070</td>
			<td>Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at &#8220;Output control&#8221; 1, &#8220;Duty cycle&#8221; 10% and time depending on the cell type used.</td>
		</tr>
		<tr>
			<td>Tabletop centrifuge for 1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>5417R&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED, N, N, N&#8242;, N&#8242;-Tetramethylethylenediamine&#160;</td>
			<td>Sigma</td>
			<td>#9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichostatin A (TSA)</td>
			<td>Sigma</td>
			<td>#T8552</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma&#160;</td>
			<td>#T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris)</td>
			<td>Sigma&#160;</td>
			<td>#T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies</td>
			<td>#15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotors&#160;</td>
			<td></td>
			<td>TLA-55, TLA-100.3 and 70.1 Ti rotors</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#357448&#160;</td>
			<td>for using with TLA-55 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#349622</td>
			<td>for using with TLA-100.3 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#355631&#160;</td>
			<td>for using with 70.1 Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuges</td>
			<td>Beckman</td>
			<td>Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent)</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath equipped with floaters or tube holders</td>
			<td></td>
			<td></td>
			<td>Set at 30&#176;C&#160;</td>
		</tr>
	</tbody>
</table>

purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles

Mikrotübül sitoskeleton çalışmalarının önemli bir yönü, in vitro rekonseptüs deneylerinde mikrotübül davranışının araştırılmasıdır. Dinamikler gibi mikrotübüllerin içsel özelliklerinin ve mikrotübül ilişkili proteinlerle (VIP' ler) etkileşimlerinin analizine izin verirler. "Tübulin kodu", mikrotübül özelliklerinin ve işlevlerinin düzenleyicileri olarak farklı tubulin izotiplerine ve çeşitli posttranslational değişikliklere (PTM' ler) işaret eden gelişmekte olan bir kavramdır. Tübulin kodunun moleküler mekanizmalarını keşfetmek için, belirli izotiplere ve PTM'lere sahip saflaştırılmış tubulin kullanarak in vitro rekonste deneyleri yapmak çok önemlidir.
Bugüne kadar, in vitro deneylerde yaygın olarak kullanılan, birçok PTM'yi barındıran ve tanımlanmış bir izotip bileşimine sahip olan beyin tübulini teknik olarak zordu. Bu nedenle, tübulini farklı kaynaklardan ve farklı izotip bileşimleri ve kontrollü PTM'lerden arındırmak için, polimerizasyon ve depolimerizasyon döngülerinin klasik yaklaşımını kullanarak bu protokolü geliştirdik. Benzeşim saflaştırmasına dayanan mevcut yöntemlerle karşılaştırıldığında, bu yaklaşım saf, polimerizasyona yetkin tübulin verir, çünkü polimerizasyona veya depolimerizasyona dirençli tübulin ardışık saflaştırma adımları sırasında atılır.
Tübulinin hücre çizgilerinden arındırılmasını, süspansiyonda veya bağlı kültürler olarak ve tek fare beyinlerinden tanımlarız. Yöntem ilk olarak hem süspansiyon hem de yapışık ayarlarda hücre kütlesinin neslini, lizis adımını, ardından polimerizasyon-depolimerizasyon döngüleri ile tübulin saflaştırmanın ardışık aşamalarını açıklar. Yöntemimiz, tübulin kodunun mikrotübüllerin içsel özellikleri ve ilişkili proteinlerle mikrotübül etkileşimleri üzerindeki etkisini ele alan deneylerde kullanılabilecek tübulin verir.

Bu yöntemin temel amacı, saflaştırılmış bileşenlerle tekrarlanan in vitro deneyler yapmak için yeterli miktarlarda yüksek kaliteli, montaja yetkin tubulin üretmektir. Bu tübulinden monte edilen mikrotübüller, dinamik veya kararlı mikrotübüller ile toplam iç yansıma floresan (TIRF) mikroskopi tekniğine dayalı rekonstrüt tahlillerinde, mikrotübül dinamiklerini test eden deneylerde, MAP'ler veya moleküler motorlarla etkileşimlerde ve motorlar tarafından kuvvet üretiminde kullanılabilir<sup class...

Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Bu protokol, polimerizasyon ve depolimerizasyon döngüleri kullanılarak kültürlü hücreler veya tek fare beyinleri gibi küçük/orta ölçekli kaynaklardan tübulin saflaştırmayı açıklar. Saflaştırılmış tubulin belirli izotiplerde zenginleştirilmiştir veya spesifik posttranslational modifikasyonlara sahiptir ve mikrotübül dinamiklerini ve etkileşimlerini incelemek için in vitro rekonsiyel testlerinde kullanılabilir.

Tubulin'in Kontrollü Posttranslational Modifikasyonlar ve Sınırlı Kaynaklardan İzotiplerle Polimerizasyon-Depolimerizasyon Döngüleri ile Saflaştırılması

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

purification-tubulin-with-controlled-posttranslational-modifications

Research

JoVE Journal

Biochemistry

5.0K Görüntüleme.  Institut Curie. Bu protokol, polimerizasyon ve depolimerizasyon döngüleri kullanılarak kültürlü hücreler veya tek fare beyinleri gibi küçük/orta ölçekli kaynaklardan tübulin saflaştırmayı açıklar. Saflaştırılmış tubulin belirli izotiplerde zenginleştirilmiştir veya spesifik posttranslational modifikasyonlara sahiptir ve mikrotübül dinamiklerini ve etkileşimlerini incelemek için in vitro rekonsiyel testlerinde kullanılabilir.

Video: Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Ökaryotik Hücrelerde Protein Kompleksleri Tandem Affinity Arıtma

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Biyokimya

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential Scanning Calorimetry &amp;#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Diferansiyel Taramalı Kalorimetre - Protein Antigen Termal İstikrar ve Yapısının Değerlendirilmesi Bir Yöntem

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Yaşlanmaya Bağlı Translasyon Sonrası Değişiklikleri İncelemek İçin Birden Fazla Fare Dokusundan Ara Filament Proteinlerinin İzolasyonu

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

Arıtma tetkikine ve fare karaciğer sinüsoidal endotel hücrelerinden perfüzyon

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin ve Ubiquitin benzeri Bağımlı Post-translational Modifikasyonlar ve Önemli Değişikliklerin Belirlenmesi Profilleme

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

U937 Hücrelerinin İzopiknik Yoğunluk Degrade Saflaştırması ile Hücre Fraksiyonasyonu

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

FAHD1 Proteininin Domuz Böbreği ve Fare Karaciğerinden Ekstraksiyonu ve Saflaştırılması

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Memeli Hücrelerinden Ubikitinlenmiş p53 Proteinlerinin Saflaştırılması

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

Her Yerde Bulunan, SUMOile Edilmiş ve ADP-Ribosile Edilmiş DNA-Protein Çapraz Bağlarının Ölçülmesi

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Yazar Spotlight: DNA Protein Çapraz Bağlarının Kantitatif Tespiti ve Translasyon Sonrası Modifikasyonları  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-Protein Çapraz Bağlarının Kantitatif Tespiti ve Translasyon Sonrası Modifikasyonları

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...