Diese Arbeit wurde unterstützt durch den ANR-10-IDEX-0001-02, den LabEx Cell'n'Scale ANR-11-LBX-0038 und das Institut de convergence Q-life ANR-17-CONV-0005. Cj wird vom Institut Curie, der französischen National Research Agency (ANR) vergeben ANR-12-BSV2-0007 und ANR-17-CE13-0021, dem Institut National du Cancer (INCA) Grant 2014-PL BIO-11-ICR-1 und der Fondation pour la Recherche Medicale (FRM) Grant DEQ2017036756. MMM wird durch das Fondation Vaincre Alzheimer Stipendium FR-16055p und durch das France Alzheimer Grant AAP SM 2019 n°2023 unterstützt. JAS wurde durch das Forschungs- und Innovationsprogramm Horizont 2020 der Europäischen Union im Rahmen der Marie-Skodowska-Curie-Zuschussvereinbarung Nr. 675737 und des FRM-Zuschusses FDT201904008210 unterstützt. SB wurde durch den FRM-Zuschuss FDT201805005465 unterstützt.
Wir danken allen Mitgliedern des Janke-Labors, insbesondere J. Souphron, sowie G. Lakisic (Institut MICALIS, AgroParisTech) und A. Gautreau (Ecole Polytechnique) für die Hilfe bei der Erstellung des Protokolls. Wir danken der Tieranlage des Institut Curie für die Hilfe bei der Mauszucht und -pflege.
Der von J. Frankel und M. Nelson entwickelte Antikörper 12G10 wurde von der Developmental Studies Hybridoma Bank gewonnen, die unter der Schirmherrschaft der NICHD entwickelt und von der University of Iowa gepflegt wurde.

<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. M. <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+and+its+role+in+controlling+microtubule+properties+and+functions.">The tubulin code and its role in controlling microtubule properties and functions.</a> Nature Reviews: Molecular Cell Biology. 21 (6), 307-326 (2020).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312 (5991), 237-242 (1984).</li><li>Margolis, R. L., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+end+assembly+and+disassembly+of+microtubules+at+steady+state+in+vitro.">Opposite end assembly and disassembly of microtubules at steady state in vitro.</a> Cell. 13 (1), 1-8 (1978).</li><li>Borisy, G. G., Olmsted, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleated+assembly+of+microtubules+in+porcine+brain+extracts.">Nucleated assembly of microtubules in porcine brain extracts.</a> Science. 177 (55), 1196-1197 (1972).</li><li>Kirschner, M. W., Williams, 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=The+mechanism+of+microtubule+assembly+in+vitro.">The mechanism of microtubule assembly in vitro.</a> Journal of Supramolecular Structure. 2 (2-4), 412-428 (1974).</li><li>Baas, P. W., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooks+and+comets:+The+story+of+microtubule+polarity+orientation+in+the+neuron.">Hooks and comets: The story of microtubule polarity orientation in the neuron.</a> Developmental Neurobiology. 71 (6), 403-418 (2011).</li><li>Stepanova, T., 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=Visualization+of+microtubule+growth+in+cultured+neurons+via+the+use+of+EB3-GFP+(end-binding+protein+3-green+fluorescent+protein).">Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).</a> Journal of Neuroscience. 23 (7), 2655-2664 (2003).</li><li>Nedelec, F. J., Surrey, T., Maggs, A. C., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+microtubules+and+motors.">Self-organization of microtubules and motors.</a> Nature. 389 (6648), 305-308 (1997).</li><li>Bieling, P., Telley, I. A., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimal+midzone+protein+module+controls+formation+and+length+of+antiparallel+microtubule+overlaps.">A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps.</a> Cell. 142 (3), 420-432 (2010).</li><li>Roostalu, 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=Directional+switching+of+the+kinesin+Cin8+through+motor+coupling.">Directional switching of the kinesin Cin8 through motor coupling.</a> Science. 332 (6025), 94-99 (2011).</li><li>Schaedel, L., 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=Microtubules+self-repair+in+response+to+mechanical+stress.">Microtubules self-repair in response to mechanical stress.</a> Nature Materials. 14 (11), 1156-1163 (2015).</li><li>Hendricks, A. G., Goldman, Y. E., Holzbaur, E. L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+the+motility+of+isolated+intracellular+cargoes.">Reconstituting the motility of isolated intracellular cargoes.</a> Methods in Enzymology. 540, 249-262 (2014).</li><li>Dogterom, M., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+organization+in+vitro.">Microtubule organization in vitro.</a> Current Opinion in Cell Biology. 25 (1), 23-29 (2013).</li><li>Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+assembly+purification+of+microtubules+without+assembly-promoting+agents+and+further+purification+of+tubulin,+microtubule-associated+proteins,+and+MAP+fragments.">Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments.</a> Methods in Enzymology. 134, 89-104 (1986).</li><li>Castoldi, M., Popov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+brain+tubulin+through+two+cycles+of+polymerization-depolymerization+in+a+high-molarity+buffer.">Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer.</a> Protein Expression and Purification. 32 (1), 83-88 (2003).</li><li>Widlund, P. O., 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=One-step+purification+of+assembly-competent+tubulin+from+diverse+eukaryotic+sources.">One-step purification of assembly-competent tubulin from diverse eukaryotic sources.</a> Molecular Biology of the Cell. 23 (22), 4393-4401 (2012).</li><li>Minoura, I., 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=Overexpression,+purification,+and+functional+analysis+of+recombinant+human+tubulin+dimer.">Overexpression, purification, and functional analysis of recombinant human tubulin dimer.</a> FEBS Letters. 587 (21), 3450-3455 (2013).</li><li>Uchimura, S., 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=A+flipped+ion+pair+at+the+dynein-microtubule+interface+is+critical+for+dynein+motility+and+ATPase+activation.">A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.</a> Journal of Cell Biology. 208 (2), 211-222 (2015).</li><li>Pamula, M. C., Ti, S. C., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structured+core+of+human+beta+tubulin+confers+isotype-specific+polymerization+properties.">The structured core of human beta tubulin confers isotype-specific polymerization properties.</a> Journal of Cell Biology. 213 (4), 425-433 (2016).</li><li>Vemu, A., 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=Structure+and+dynamics+of+single-isoform+recombinant+neuronal+Human+tubulin.">Structure and dynamics of single-isoform recombinant neuronal Human tubulin.</a> Journal of Biological Chemistry. 291 (25), 12907-12915 (2016).</li><li>Ti, S. C., Alushin, G. M., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+beta-tubulin+isotypes+can+regulate+microtubule+protofilament+number+and+stability.">Human beta-tubulin isotypes can regulate microtubule protofilament number and stability.</a> Developmental Cell. 47 (2), 175-190 (2018).</li><li>Souphron, 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=Purification+of+tubulin+with+controlled+post-translational+modifications+by+polymerization-depolymerization+cycles.">Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.</a> Nature Protocols. 14, 1634-1660 (2019).</li><li>Barisic, M., 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=Microtubule+detyrosination+guides+chromosomes+during+mitosis.">Microtubule detyrosination guides chromosomes during mitosis.</a> Science. 348 (6236), 799-803 (2015).</li><li>Nirschl, J. J., Magiera, M. M., Lazarus, J. E., Janke, C., Holzbaur, E. L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Tubulin+tyrosination+and+CLIP-170+phosphorylation+regulate+the+initiation+of+dynein-driven+transport+in+neurons.">alpha-Tubulin tyrosination and CLIP-170 phosphorylation regulate the initiation of dynein-driven transport in neurons.</a> Cell Reports. 14 (11), 2637-2652 (2016).</li><li>Guedes-Dias, P., 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=Kinesin-3+responds+to+local+microtubule+dynamics+to+target+synaptic+cargo+delivery+to+the+presynapse.">Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse.</a> Current Biology. 29 (2), 268-282 (2019).</li><li>Luo, Y., 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=Direct+observation+of+dynamic+protein+interactions+involving+human+microtubules+using+solid-state+NMR+spectroscopy.">Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.</a> Nature Communications. 11 (1), 18 (2020).</li><li>Even, A., 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=ATAT1-enriched+vesicles+promote+microtubule+acetylation+via+axonal+transport.">ATAT1-enriched vesicles promote microtubule acetylation via axonal transport.</a> Science Advances. 5 (12), 2705 (2019).</li><li>Wolff, J., Sackett, D. L., Knipling, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation+selective+promotion+of+tubulin+polymerization+by+alkali+metal+chlorides.">Cation selective promotion of tubulin polymerization by alkali metal chlorides.</a> Protein Science. 5 (10), 2020-2028 (1996).</li><li>Magiera, M. M., 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=Excessive+tubulin+polyglutamylation+causes+neurodegeneration+and+perturbs+neuronal+transport.">Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.</a> EMBO Journal. 37 (23), 100440 (2018).</li><li>Lacroix, B., 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=Generation+of+differentially+polyglutamylated+microtubules.">Generation of differentially polyglutamylated microtubules.</a> Methods in Molecular Biology. 777, 57-69 (2011).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Magiera, M. M., Janke, C., Correia, J. J., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology Vol. 115 Microtubules, in vitro. , 247-267 (2013).</li><li>Hausrat, T. J., Radwitz, J., Lombino, F. L., Breiden, P., Kneussel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-+and+beta-tubulin+isotypes+are+differentially+expressed+during+brain+development.">Alpha- and beta-tubulin isotypes are differentially expressed during brain development.</a> Developmental Neurobiology. , (2020).</li><li>Janke, C., 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=Tubulin+polyglutamylase+enzymes+are+members+of+the+TTL+domain+protein+family.">Tubulin polyglutamylase enzymes are members of the TTL domain protein family.</a> Science. 308 (5729), 1758-1762 (2005).</li><li>Newton, C. N., 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=Intrinsically+slow+dynamic+instability+of+HeLa+cell+microtubules+in+vitro.">Intrinsically slow dynamic instability of HeLa cell microtubules in vitro.</a> Journal of Biological Chemistry. 277 (45), 42456-42462 (2002).</li><li>Belvindrah, R., 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=Mutation+of+the+alpha-tubulin+Tuba1a+leads+to+straighter+microtubules+and+perturbs+neuronal+migration.">Mutation of the alpha-tubulin Tuba1a leads to straighter microtubules and perturbs neuronal migration.</a> Journal of Cell Biology. 216 (8), 2443-2461 (2017).</li><li>Breuss, M., 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=Mutations+in+the+murine+homologue+of+TUBB5+cause+microcephaly+by+perturbing+cell+cycle+progression+and+inducing+p53+associated+apoptosis.">Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53 associated apoptosis.</a> Development. , (2016).</li><li>Latremoliere, A., 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=Neuronal-specific+TUBB3+is+not+required+for+normal+neuronal+function+but+is+essential+for+timely+axon+regeneration.">Neuronal-specific TUBB3 is not required for normal neuronal function but is essential for timely axon regeneration.</a> Cell Reports. 24 (7), 1865-1879 (2018).</li><li>Morley, S. 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>

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Die hier beschriebene Methode bietet eine Plattform, um schnell hochwertiges, montagekompetentes Tubulin in mittelgroßen Mengen aus Zelllinien und einzelnen Mausgehirnen zu erzeugen. Es basiert auf dem Gold-Standard-Protokoll der Tubulinreinigung von Rinderhirnen, die seit vielen Jahren im Feld verwendet werden16,17. Ein besonderer Vorteil des Ansatzes ist die Verwendung von Suspensionskulturen von HeLa S3-Zellen, die, sobald sie etabliert sind, große Mengen an...

Mikrotubuli spielen in vielen zellulären Prozessen eine entscheidende Rolle. Sie geben Zellen ihre Form, bauen meiotische und mitotische Spindeln für die Chromosomensegregation und dienen als Spuren für den intrazellulären Transport. Um diese vielfältigen Funktionen auszuführen, organisieren sich Mikrotubuli auf unterschiedliche Weise. Eine der faszinierendsten Fragen auf diesem Gebiet ist es, die molekularen Mechanismen zu verstehen, die es den strukturell und evolutionär konservierten Mikrotubuli ermöglichen, sich an diese Fülle von Organisationen und Funktionen anzupassen. Ein potenzieller Mechanismus ist die Diversifizierung der Mikrotubuli, die durch das Konzept definiert wird, das als "Tubulin-Code"1,2,3" bekannt ist. Der Tubulin-Code umfasst zwei Hauptkomponenten: die Differentialintegration von α- und β-Tubulin-Genprodukten (Tubulin-Isotypen) in die Mikrotubuli und Tubulin-Posttranslationalmodifikationen (PTMs).
Seit den 1970er Jahren haben In-vitro-Rekonstitutionsexperimente, kombiniert mit sich entwickelnden Lichtmikroskopie-Techniken, den Weg für wichtige Entdeckungen über die Eigenschaften von Mikrotubuli geebnet: dynamische Instabilität4 und Laufband5,und ihre anderen Mechanismen und Funktionen6,7,8,9,10,11,12,13,14,15. Fast alle bisher durchgeführten In-vitro-Experimente basierten auf Tubulin, das aus Hirngewebe mit wiederholten Zyklen der Polymerisation und Depolymerisation16,17gereinigt wurde. Obwohl die Reinigung aus dem Hirngewebe den Vorteil der Gewinnung von hochwertigem Tubulin in großen Mengen (in der Regel Grammmengen) verleiht, ist ein wichtiger Nachteil die Heterogenität, da Tubulin, das aus Hirngewebe gereinigt wird, eine Mischung aus verschiedenen Tubulin-Isotypen ist und mit vielen Tubulin-PTMs angereichert ist. Diese Heterogenität macht es unmöglich, die Rolle eines bestimmten Tubulin-PTM oder Isotyps bei der Kontrolle von Mikrotubuli-Eigenschaften und -Funktionen abzustecken. Daher ist die Herstellung von Montage-kompetentem Tubulin mit kontrollierten Tubulin-PTMs und homogener Isotypzusammensetzung unerlässlich, um die molekularen Mechanismen des Tubulincodes anzugehen.
Kürzlich wurde ein Ansatz zur Reinigung von Tubulin durch Affinitätschromatographie mit der mikrotubulienbindenden TOG-Domäne (Tumor-überexprimiertes Gen) der Hefe Stu2pentwickelt. Bei dieser Methode wird Tubulin in rohen Lysaten von Zellen oder Geweben durch eine Säule geleitet, wo es an die matriximmobilisierte TOG-Domäne bindet, die die Analyse des gesamten Tubulinpools einer bestimmten, sogar sehr kleinen Probe ermöglicht. Ein lang erwarteter Ansatz zur Reinigung des rekombinanten Tubulins wurde in den letzten Jahren ebenfalls beschrieben. Es basiert auf dem Baculovirus-System, in dem ein bi-cistronischer Vektor, der α- und β-Tubulin-Gene enthält, in Insektenzellen19exprimiert wird. Diese Methode ist jedoch sehr umständlich und zeitaufwändig und wird daher hauptsächlich zur Untersuchung der Auswirkungen der Tubulinmutationen20 und der Tubulin-Isotypen21,22,23 in vitro verwendet.
Im aktuellen Protokoll beschreiben wir eine Methode, die den etablierten und weit verbreiteten Polymerisations-Depolymerisationsansatz als Blaupause verwendet, um Tubulin mit unterschiedlichen Modifikationsstufen entweder aus Zelllinien oder aus Maushirngewebe zu erzeugen24. Bei diesem Verfahren wird Tubulin zwischen dem löslichen (Tubulin-Dimer bei 4 °C) und der polymerisierten Form (Mikrotubuli bei 30 °C in Gegenwart von Guanosin 5'-Triphosphat [GTP]) geradelt. Jede Form wird durch aufeinander folgende Zentrifugationsschritte getrennt: Tubulin-Dimere bleiben nach einer Erkältung (4 °C) im Überstand, während Mikrotubuli bei 30 °C pelletiert werden. Darüber hinaus wird ein Polymerisationsschritt bei hoherPiperazin-N-Bis(2-Ethansulfonsäure) (PIPES) Konzentration durchgeführt, die die Entfernung von mikrotubuli-assoziierten Proteinen aus den Mikrotubuli und damit aus dem schließlich gereinigten Tubuli ermöglicht. Tubulin gereinigt aus HeLa S3 Zellen als Suspension oder anhantibe Kulturen gewachsen ist praktisch frei von jedem Tubulin PTM und wurde in den jüngsten In-vitro-Rekonstitutionsexperimente25,26,27,28verwendet. Wir haben die Methode zur Reinigung von Tubulin von einzelnen Mausgehirnen weiter angepasst, die für eine große Anzahl von Mausmodellen mit Änderungen in Tubulin-Isotypen und PTMs verwendet werden können.
Im Protokoll beschreiben wir zunächst die Erzeugung des Ausgangsmaterials (Zellmasse oder Hirngewebe), seine Lyse (Abbildung 1A), gefolgt von den aufeinanderfolgenden Schritten der Tubulinpolymerisation und Depolymerisation zur Reinigung des Tubulins (Abbildung 1B). Wir beschreiben ferner das Verfahren zur Beurteilung der Reinheit (Abbildung 2A,B) und der Menge (Abbildung 3A,B) des gereinigten Tubulins. Das Verfahren kann angepasst werden, um Tubulin zu produzieren, das mit einem ausgewählten PTM angereichert ist, indem ein modifizierendes Enzym in Zellen vor der Tubulinreinigung überexzessiert wird (Abbildung 4B). Alternativ können Tubulin-modifizierende Enzyme während des Reinigungsprozesses dem Tubulizuz hinzugefügt werden. Schließlich können wir Tubulin ohne spezifische Isotypen oder PTMs aus dem Gehirn von Mäusen reinigen, die einen Mangel an den entsprechenden Tubulin-modifizierenden Enzymen aufweisen (Abbildung 4B)29.
Die Methode, die wir hier beschreiben, hat zwei Hauptvorteile: (i) es ermöglicht die Produktion ausreichend großer Mengen Tubulin in relativ kurzer Zeit, und (ii) es erzeugt hochwertiges, reines Tubulin, entweder mit spezifischer Tubulin-Isotypzusammensetzung oder PTMs. Im zugehörigen Video dieses Manuskripts heben wir einige der kritischen Schritte dieses Verfahrens hervor.

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			<td>1&#160;M MgCl2&#160;</td>
			<td>Sigma</td>
			<td>#M1028</td>
			<td></td>
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			<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

Ein wichtiger Aspekt der Untersuchungen des Mikrotubuli-Zytoskeletts ist die Untersuchung des Mikrotubuli-Verhaltens in In-vitro-Rekonstitutionsexperimenten. Sie ermöglichen die Analyse der intrinsischen Eigenschaften von Mikrotubuli, wie dynamik, und ihrer Wechselwirkungen mit mikrotubuli-assoziierten Proteinen (MAPs). Der "Tubulin-Code" ist ein neu entstehendes Konzept, das auf verschiedene Tubulin-Isotypen und verschiedene posttranslationale Modifikationen (PTMs) als Regulatoren von Mikrotubuli-Eigenschaften und -Funktionen verweist. Um die molekularen Mechanismen des Tubulin-Codes zu erforschen, ist es entscheidend, In-vitro-Rekonstitutionsexperimente mit gereinigtem Tubulin mit spezifischen Isotypen und PTMs durchzuführen.
Bis heute war dies technisch eine Herausforderung als Gehirn Tubulin, das weit verbreitet in In-vitro-Experimente verwendet wird, beherbergt viele PTMs und hat eine definierte Isotypzusammensetzung. Daher haben wir dieses Protokoll entwickelt, um Tubulin aus verschiedenen Quellen und mit unterschiedlichen Isotypzusammensetzungen und kontrollierten PTMs zu reinigen, wobei der klassische Ansatz von Polymerisations- und Depolymerisationszyklen verwendet wird. Im Vergleich zu bestehenden Methoden, die auf der Affinitätsreinigung basieren, ergibt dieser Ansatz reines, polymerisationskompetentes Tubulin, da Tubulin, das gegen Polymerisation oder Depolymerisation resistent ist, während der aufeinanderfolgenden Reinigungsschritte verworfen wird.
Wir beschreiben die Reinigung von Tubulin aus Zelllinien, die entweder in Suspension oder als anhantibe Kulturen angebaut werden, und von einzelnen Mausgehirnen. Das Verfahren beschreibt zunächst die Erzeugung von Zellmasse sowohl in Suspensions- als auch in Hafteinstellungen, dem Lyseschritt, gefolgt von den aufeinanderfolgenden Stadien der Tubulinreinigung durch Polymerisations-Depolymerisationszyklen. Unsere Methode liefert Tubulin, das in Experimenten verwendet werden kann, die sich mit dem Einfluss des Tubulin-Codes auf die intrinsischen Eigenschaften von Mikrotubuli und Mikrotubuli-Wechselwirkungen mit assoziierten Proteinen befassen.

Das Hauptziel dieser Methode ist die Herstellung von hochwertigem, montagekompetentem Tubulin in Mengen, die für wiederholte In-vitro-Experimente mit den gereinigten Komponenten ausreichen. Aus diesem Tubulin zusammengesetzte Mikrotubuli können in Rekonstitutionstests verwendet werden, die auf der gesamten internen Reflexionsfluoreszenz-Mikroskopie (TIRF) mit dynamischen oder stabilen Mikrotubuli basieren, in Experimenten, die Mikrotubulidynamik, Wechselwirkungen mit MAPs oder molekularen Motoren und die Krafterzeugung...

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

Dieses Protokoll beschreibt die Tubulinreinigung aus kleinen/mittleren Quellen wie kultivierten Zellen oder Gehirnen mit einer Maus unter Verwendung von Polymerisations- und Depolymerisationszyklen. Das gereinigte Tubulin ist in bestimmten Isotypen angereichert oder hat spezifische posttranslationale Modifikationen und kann in In-vitro-Rekonstitutions-Assays zur Untersuchung der Mikrotubuli-Dynamik und -Wechselwirkungen verwendet werden.

Reinigung von Tubulin mit kontrollierten posttranslationalen Modifikationen und Isotypen aus begrenzten Quellen durch Polymerisations-Depolymerisationszyklen

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 Aufrufe.  Institut Curie. Dieses Protokoll beschreibt die Tubulinreinigung aus kleinen/mittleren Quellen wie kultivierten Zellen oder Gehirnen mit einer Maus unter Verwendung von Polymerisations- und Depolymerisationszyklen. Das gereinigte Tubulin ist in bestimmten Isotypen angereichert oder hat spezifische posttranslationale Modifikationen und kann in In-vitro-Rekonstitutions-Assays zur Untersuchung der Mikrotubuli-Dynamik und -Wechselwirkungen verwendet werden.

Serie: 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.

Tandem Affinity Purification von Proteinkomplexen aus eukaryotischen Zellen

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

Forschung

Biochemie

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.

Differential Scanning Calorimetry - Eine Methode zur Beurteilung der thermischen Stabilität und Konformation von 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 ...

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.

Isolierung von Zwischenfilamentproteinen aus mehreren Mausgeweben zur Untersuchung von altern-assoziierten posttranslationalen Modifikationen

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.

Reinigung von Hepatozyten und sinusförmigen Endothelzellen aus Maus Leber Perfusion

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.

Profilierung von Ubiquitin und Ubiquitin-ähnlichen abhängigen posttranslationalen Änderungen und Identifizierung signifikanter Änderungen

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.

Zellfraktionierung von U937-Zellen durch isopyknische Dichtegradientenreinigung

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.

Extraktion und Reinigung von FAHD1-Protein aus Schweineniere und Mausleber

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.

Aufreinigung ubiquitinierter p53-Proteine aus Säugetierzellen

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

Messung von ubiquitylierten, SUMOylierten und ADP-ribosylierten DNA-Protein-Vernetzungen

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.

Autor im Rampenlicht: Quantitativer Nachweis von DNA-Protein-Vernetzungen und deren posttranslationale Modifikationen  

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.

Quantitativer Nachweis von DNA-Protein-Vernetzungen und deren posttranslationale Modifikationen

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