Name: quantitative microtubule fractionation technique to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues
Uploaded: 2023-11-17T13:50:00
Description: Watch this Scientific Journal Video about Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues at JoVE.com

这项工作在一定程度上得到了JST的支持，建立了大学奖学金，以创造科学技术创新（A.HT。JPMJFS2145）、JST SPRING（A.HT.;JPMJSP2129）、JSPS 研究员补助金 （A.HT.; 23KJ2078）、科学研究补助金 （B） JSPS KAKENHI （22H02946 for TM）、文部科学省 （TM; 26117004） 的名为“脑蛋白衰老和痴呆症控制”的创新领域科学研究补助金，以及上原纪念基金会 （TM; 202020027） 的上原研究奖学金。作者声明没有相互竞争的经济利益。

1.MT分馏法
注：本研究中进行的所有实验均已获得同志社大学动物伦理委员会的批准。这里使用3-4个月大的C57BL / 6J小鼠。在该方案中，解剖的组织，例如脑，肝脏或胸腺，立即在冰冷的微管稳定缓冲液（MSB）中匀浆，其含有紫杉醇（MT稳定剂）的浓度，其浓度不仅阻止了解聚，而且阻止了MT的再聚合。通过两步超速离心过程将匀浆分离成三部分（<strong class="xfig" style...

<ol>
	<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>Conde, C., Caceres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+assembly,+organization+and+dynamics+in+axons+and+dendrites.">Microtubule assembly, organization and dynamics in axons and dendrites.</a> Nature Reviews Neuroscience. 10 (5), 319-332 (2009).</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>Baas, P. W., Rao, A. N., Matamoros, A. J., Leo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+properties+of+neuronal+microtubules.">Stability properties of neuronal microtubules.</a> Cytoskeleton (Hoboken). 73 (9), 442-460 (2016).</li><li>Challacombe, J. F., Snow, D. M., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+microtubule+ends+are+required+for+growth+cone+turning+to+avoid+an+inhibitory+guidance+cue.">Dynamic microtubule ends are required for growth cone turning to avoid an inhibitory guidance cue.</a> Journal of Neuroscience. 17 (9), 3085-3095 (1997).</li><li>Kapitein, L. C., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+neuronal+microtubule+cytoskeleton.">Building the neuronal microtubule cytoskeleton.</a> Neuron. 87 (3), 492-506 (2015).</li><li>Leo, 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=Vertebrate+fidgetin+restrains+axonal+growth+by+severing+labile+domains+of+microtubules.">Vertebrate fidgetin restrains axonal growth by severing labile domains of microtubules.</a> Cell Reports. 12 (11), 1723-1730 (2015).</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>Wloga, D., Joachimiak, E., Fabczak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin+post-translational+modifications+and+microtubule+dynamics.">Tubulin post-translational modifications and microtubule dynamics.</a> International Journal of Molecular Sciences. 18 (10), 2207 (2017).</li><li>Baas, P. W., Black, 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=Individual+microtubules+in+the+axon+consist+of+domains+that+differ+in+both+composition+and+stability.">Individual microtubules in the axon consist of domains that differ in both composition and stability.</a> Journal of Cell Biology. 111 (2), 495-509 (1990).</li><li>Cartelli, D., 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+alterations+occur+early+in+experimental+parkinsonism+and+the+microtubule+stabilizer+epothilone+D+is+neuroprotective.">Microtubule alterations occur early in experimental parkinsonism and the microtubule stabilizer epothilone D is neuroprotective.</a> Scientific Reports. 3, 1837 (2013).</li><li>Zhang, B., 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-binding+drugs+offset+tau+sequestration+by+stabilizing+microtubules+and+reversing+fast+axonal+transport+deficits+in+a+tauopathy+model.">Microtubule-binding drugs offset tau sequestration by stabilizing microtubules and reversing fast axonal transport deficits in a tauopathy model.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (1), 227-231 (2005).</li><li>Zhang, F., 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=Post-translational+modifications+of+alpha-tubulin+in+Alzheimer+disease.">Post-translational modifications of alpha-tubulin in Alzheimer disease.</a> Translational Neurodegeneration. 4, 9 (2015).</li><li>Miyasaka, 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=Curcumin+improves+tau-induced+neuronal+dysfunction+of+nematodes.">Curcumin improves tau-induced neuronal dysfunction of nematodes.</a> Neurobiology of Aging. 39, 69-81 (2016).</li><li>Fujiwara, H., 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=Inhibition+of+microtubule+assembly+competent+tubulin+synthesis+leads+to+accumulation+of+phosphorylated+tau+in+neuronal+cell+bodies.">Inhibition of microtubule assembly competent tubulin synthesis leads to accumulation of phosphorylated tau in neuronal cell bodies.</a> Biochemical and Biophysical Research Communications. 521 (3), 779-785 (2020).</li><li>Vielkind, U., Swierenga, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+fixation+procedure+for+immunofluorescent+detection+of+different+cytoskeletal+components+within+the+same+cell.">A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell.</a> Histochemistry. 91 (1), 81-88 (1989).</li><li>Kanai, 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=Expression+of+multiple+tau+isoforms+and+microtubule+bundle+formation+in+fibroblasts+transfected+with+a+single+tau+cDNA.">Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.</a> Journal of Cell Biology. 109 (3), 1173-1184 (1989).</li><li>Brown, A., Li, Y., Slaughter, T., Black, 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=Composite+microtubules+of+the+axon:+quantitative+analysis+of+tyrosinated+and+acetylated+tubulin+along+individual+axonal+microtubules.">Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules.</a> Journal of Cell Science. 104 (2), 339-352 (1993).</li><li>Black, M. M., Slaughter, T., Moshiach, S., Obrocka, M., Fischer, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+is+enriched+on+dynamic+microtubules+in+the+distal+region+of+growing+axons.">Tau is enriched on dynamic microtubules in the distal region of growing axons.</a> Journal of Neuroscience. 16 (11), 3601-3619 (1996).</li><li>Caron, J. M., Jones, A. L., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+of+tubulin+synthesis+in+hepatocytes+and+fibroblasts.">Autoregulation of tubulin synthesis in hepatocytes and fibroblasts.</a> Journal of Cell Biology. 101 (5), 1763-1772 (1985).</li><li>Merrick, S. E., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+destruction+of+stable+microtubules+and+axons+by+inhibitors+of+protein+serine/threonine+phosphatases+in+cultured+human+neurons.">Selective destruction of stable microtubules and axons by inhibitors of protein serine/threonine phosphatases in cultured human neurons.</a> Journal of Neuroscience. 17 (15), 5726-5737 (1997).</li><li>Miyasaka, T., Sato, S., Tatebayashi, Y., Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+destruction+induces+tau+liberation+and+its+subsequent+phosphorylation.">Microtubule destruction induces tau liberation and its subsequent phosphorylation.</a> FEBS Letters. 584 (14), 3227-3232 (2010).</li><li>Hagita, 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=Quantitative+fractionation+of+tissue+microtubules+with+distinct+biochemical+properties+reflecting+their+stability+and+lability.">Quantitative fractionation of tissue microtubules with distinct biochemical properties reflecting their stability and lability.</a> Biochemical and Biophysical Research Communications. 560, 186-191 (2021).</li><li>Montecinos-Franjola, F., Chaturvedi, S. K., Schuck, P., Sackett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+tubulins+are+not+alike:+Heterodimer+dissociation+differs+among+different+biological+sources.">All tubulins are not alike: Heterodimer dissociation differs among different biological sources.</a> Journal of Biological Chemistry. 294 (26), 10315-10324 (2019).</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=A+taxol-dependent+procedure+for+the+isolation+of+microtubules+and+microtubule-associated+proteins+(MAPs).">A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs).</a> Journal of Cell Biology. 92 (2), 435-442 (1982).</li><li>Bartolo, M. E., Carter, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+microtubule+stabilization+on+the+freezing+tolerance+of+mesophyll+cells+of+spinach.">Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach.</a> Plant Physiology. 97 (1), 182-187 (1991).</li><li>Strang, K. H., Golde, T. E., Giasson, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPT+mutations,+tauopathy,+and+mechanisms+of+neurodegeneration.">MAPT mutations, tauopathy, and mechanisms of neurodegeneration.</a> Laboratory Investigation. 99 (7), 912-928 (2019).</li><li>Fourel, G., Boscheron, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin+mutations+in+neurodevelopmental+disorders+as+a+tool+to+decipher+microtubule+function.">Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function.</a> FEBS Letters. 594 (21), 3409-3438 (2020).</li><li>Terry, R. D., Gonatas, N. K., Weiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+in+Alzheimer's+presenile+dementia.">Ultrastructural studies in Alzheimer's presenile dementia.</a> The American Journal of Pathology. 44 (2), 269-297 (1964).</li><li>Yoshida, H., Ihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+in+paired+helical+filaments+is+functionally+distinct+from+fetal+tau:+assembly+incompetence+of+paired+helical+filament-tau.">Tau in paired helical filaments is functionally distinct from fetal tau: assembly incompetence of paired helical filament-tau.</a> Journal of Neurochemistry. 61 (3), 1183-1186 (1993).</li><li>Cash, A. D., 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+reduction+in+Alzheimer's+disease+and+aging+is+independent+of+tau+filament+formation.">Microtubule reduction in Alzheimer's disease and aging is independent of tau filament formation.</a> The American Journal of Pathology. 162 (5), 1623-1627 (2003).</li><li>Hempen, B., Brion, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+acetylated+alpha-tubulin+immunoreactivity+in+neurofibrillary+tangle-bearing+neurons+in+Alzheimer's+disease.">Reduction of acetylated alpha-tubulin immunoreactivity in neurofibrillary tangle-bearing neurons in Alzheimer's disease.</a> Journal of Neuropathology and Experimental Neurology. 55 (9), 964-972 (1996).</li><li>Miyasaka, 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=Imbalanced+expression+of+tau+and+tubulin+induces+neuronal+dysfunction+in+C.+elegans+models+of+tauopathy.">Imbalanced expression of tau and tubulin induces neuronal dysfunction in C. elegans models of tauopathy.</a> Frontiers in Neuroscience. 12, 415 (2018).</li><li>Boiarska, Z., Passarella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-targeting+agents+and+neurodegeneration.">Microtubule-targeting agents and neurodegeneration.</a> Drug Discovery Today. 26 (2), 604-615 (2021).</li></ol>

在研究微管蛋白在活生物体组织中的状态时，最重要的任务是防止制备过程中意外的MT聚合或解聚。从组织去除到匀浆和离心过程中，MTS在样品中的稳定性受MSB中紫杉醇浓度、组织与缓冲液的比例、温度等因素的影响。因此，在用20倍体积的匀浆分析小鼠组织的方案的每个步骤中优化条件。较高浓度的紫杉醇可以在 体外诱导MT聚合，即使在冷却条件下也是如此25。当分析微?...

微管 （MTs） 是细长的管状结构，由α/β-微管蛋白异二聚体亚基组成的原丝组成。它们在细胞分裂、运动、形状维持和细胞内运输等各种细胞过程中发挥着重要作用，使其成为真核细胞骨架1 的组成部分。暴露α-微管蛋白亚基的 MT 负端相对稳定，而暴露β-微管蛋白亚基的正端经历动态解聚和聚合2。这种微管蛋白二聚体在正端加成和解离的连续循环，称为动态不稳定性，导致重复的救援和灾难过程3.MT 表现出具有动态不稳定性局部变化的焦点域，包括稳定域和不稳定域4。
精确控制MT的动态不稳定性对于许多细胞功能至关重要，特别是在以复杂形态为特征的神经元中。MT 的适应性和耐久性在神经细胞的发育和正常功能中起着至关重要的作用 5,6,7。已发现 MT 的动态不稳定性与微管蛋白的各种翻译后修饰 （PTM） 有关，例如乙酰化、磷酸化、棕榈酰化、去酪氨酸化、δ 2、聚谷氨酰胺氧化和聚甘氨酰化。此外，微管相关蛋白 （MAP） 的结合是一种调节机制8。PTM（不包括乙酰化）主要发生在位于 MT 外表面的微管蛋白羧基末端区域。这些修饰在 MT 上创造了不同的表面条件，影响了它们与 MAP 的相互作用，并最终控制了 MT 的稳定性9。α-微管蛋白中羧基末端酪氨酸残基的存在表明存在动态 MT，其迅速被游离微管蛋白池取代。相反，羧基末端的去酪氨酸化和 Lys40 的乙酰化表明稳定的 MT 具有降低的动态不稳定性 9,10。
微管蛋白的PTM已被广泛用于评估MTs的动力学和稳定性的实验5,7,11,12,13,14,15。例如，在细胞培养研究中，微管蛋白可以分为两个池：游离微管蛋白池和MT池。这是通过在固定剩余的 MT15、16、17、18、19 之前通过细胞通透性释放游离微管蛋白来实现的。生化方法涉及使用化学 MT 稳定剂来保护 MT 免受灾难，从而通过离心分离 MT 和游离微管蛋白20,21,22。然而，这些程序无法区分稳定和不太稳定（不稳定）的 MT，因此无法量化 MT 或大脑等组织中的可溶性微管蛋白。因此，在生理和病理条件下评估生物体的MT稳定性已被证明具有挑战性。为了解决这一实验局限性，我们开发了一种新技术，用于精确分离小鼠组织中的 MT 和游离微管蛋白23。
这种独特的 MT 分离方法涉及在维持组织中微管蛋白状态的条件下进行组织匀浆，以及两步离心以分离稳定的 MT、不稳定的 MT 和游离微管蛋白。这种简单的程序可以应用于广泛的研究，包括生物体中 MT 和 MAP 的基础研究、与 MT 稳定性相关的健康和疾病的生理和病理分析，以及开发靶向 MT 的药物和其他疗法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 ML TUBE CASE OF 500</td>
			<td>Beckman Coulter</td>
			<td>357448</td>
			<td></td>
		</tr>
		<tr>
			<td>1A2</td>
			<td>Sigma-Aldrich</td>
			<td>T9028</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td>Nacalai Tesque</td>
			<td>02442-44</td>
			<td></td>
		</tr>
		<tr>
			<td>300 kDa ultrafiltration spin column</td>
			<td>Aproscience</td>
			<td>PT-1013</td>
			<td></td>
		</tr>
		<tr>
			<td>6-11B1</td>
			<td>Sigma-Aldrich</td>
			<td>T7451</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>AKTA prime plus</td>
			<td>Cytiva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-035-146</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>antipain</td>
			<td>Peptide Institute Inc.</td>
			<td>4062</td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Nacalai Tesque</td>
			<td>03346-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemi-Lumi One L</td>
			<td>Nacalai Tesque</td>
			<td>07880-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter system</td>
			<td>Corning</td>
			<td>430758</td>
			<td>0.22&#181;m 33.2cm&#178; Nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>DIFP</td>
			<td>Sigma-Aldrich</td>
			<td>55-91-4&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DIGITAL HOMOGENIZER</td>
			<td></td>
			<td>AS ONE</td>
			<td>HOM</td>
		</tr>
		<tr>
			<td>DM1A</td>
			<td>Sigma-Aldrich</td>
			<td>T9026</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Nacalai Tesque</td>
			<td>14128-46</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Nacalai Tesque</td>
			<td>37346-05</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroTrans W 3.3 Meter Roll</td>
			<td>Pall Corporation</td>
			<td>BSP0161</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Nacalai Tesque</td>
			<td>17018-25</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td></td>
			<td>Nacalai Tesque</td>
			<td>17450-61</td>
		</tr>
		<tr>
			<td>HIGH SPEED REFRIGERATIOED MICRO CENTRIFUGE Kitman</td>
			<td></td>
			<td>TOMY</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 200 pg column</td>
			<td>Cytiva</td>
			<td>28-9893-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Gauge Software&#160;</td>
			<td>FUJIFILUM Wako Pure Chemical Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoStar LD&#160;</td>
			<td>FUJIFILUM Wako Pure Chemical Corporation</td>
			<td>292-69903</td>
			<td></td>
		</tr>
		<tr>
			<td>KMX-1</td>
			<td>Millipore</td>
			<td>MAB3408</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>LAS-4000 luminescent image analyzer</td>
			<td>FUJIFILUM Wako Pure Chemical Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Peptide Institute Inc.</td>
			<td>43449-62</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Nacalai Tesque</td>
			<td>21003-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Nacalai Tesque</td>
			<td>32013-92</td>
			<td></td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Nacalai Tesque</td>
			<td>31420-82</td>
			<td></td>
		</tr>
		<tr>
			<td>okadaic acid</td>
			<td>LC Laboratories</td>
			<td>O-2220&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTIMA MAX-XP</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Nacalai Tesque</td>
			<td>26436-52</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Nacalai Tesque</td>
			<td>27327-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Centrifuge Tubes for TLA120.2</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (cOmplete, EDTA-free)</td>
			<td></td>
			<td>Roche</td>
			<td>5056489001</td>
		</tr>
		<tr>
			<td>Purified tubulin&#160;</td>
			<td>Cytoskeleton</td>
			<td>T240</td>
			<td></td>
		</tr>
		<tr>
			<td>QSONICA Q55</td>
			<td>QSonica</td>
			<td>Q55</td>
			<td></td>
		</tr>
		<tr>
			<td>Taxol</td>
			<td></td>
			<td>LC Laboratories</td>
			<td>P-9600</td>
		</tr>
		<tr>
			<td>TLA-120.2 rotor</td>
			<td>Beckman Coulter</td>
			<td>357656</td>
			<td></td>
		</tr>
		<tr>
			<td>TLA-55 rotor</td>
			<td>Beckman Coulter</td>
			<td>366725</td>
			<td></td>
		</tr>
		<tr>
			<td>TLCK</td>
			<td>Nacalai Tesque</td>
			<td>34219-94</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque</td>
			<td>12967-45</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative microtubule fractionation technique to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues

微管由α/β-微管蛋白二聚体组成，是真核细胞细胞骨架的重要组成部分。这些管状聚合物表现出动态不稳定性，因为微管蛋白异二聚体亚基会经历重复聚合和解聚。通过微管蛋白翻译后修饰和微管相关蛋白实现微管稳定性和动力学的精确控制，对于各种细胞功能至关重要。微管功能障碍与发病机制密切相关，包括神经退行性疾病。正在进行的研究集中在调节稳定性的微管靶向治疗剂上，为这些疾病和癌症提供了潜在的治疗选择。因此，了解微管的动态状态对于评估疾病进展和治疗效果至关重要。
传统上，微管动力学是通过粗分离或免疫测定在 体外 或培养细胞中评估的，使用靶向微管蛋白翻译后修饰的抗体。然而，使用此类程序准确分析组织中的微管蛋白状态存在挑战。在这项研究中，我们开发了一种简单而创新的微管分离方法，用于分离小鼠组织中的稳定微管、不稳定微管和游离微管蛋白。
该过程涉及以19：1的体积比在微管稳定缓冲液中匀浆解剖的小鼠组织。然后，在初始慢速离心（2,400 × g）之后，通过两步超速离心过程对匀浆进行分馏以除去碎屑。第一个超速离心步骤（100,000 × g）沉淀出稳定的微管，而所得上清液则进行第二个超速离心步骤（500,000 × g）以分馏不稳定的微管和可溶性微管蛋白二聚体。该方法测定了微管蛋白在小鼠大脑中构成稳定或不稳定微管的比例。此外，观察到微管稳定性的明显组织变化与组成细胞的增殖能力相关。这些发现突出了这种新方法在分析生理和病理条件下微管稳定性方面的巨大潜力。

通过MT分馏法定量小鼠脑中P2，P3和S3组分中的微管蛋白 通过MT分馏方法将小鼠组织中的微管蛋白分离为P2，P3和S3级分，并通过蛋白质印迹进行定量（图1A）。在小鼠脑中以100,000 × g 超速离心20 min，残留在P2组分中的MTs沉淀物占总微管蛋白的34.86%±1.68%。将上清液（S2）进一步以500,000 × g 离心60分钟。获得沉淀物（P3级分）和上清液（S3级分），分别占...

Watch this Scientific Journal Video about Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues at JoVE.com

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

微管是微管蛋白聚合物，在真核细胞中作为细胞骨架成分起着至关重要的作用，并以其动态不稳定性而闻名。本研究开发了一种将微管分离为稳定微管、不稳定微管和游离微管蛋白的方法，以评估微管在各种小鼠组织中的稳定性。

定量微管分离技术分离小鼠组织中稳定的微管、不稳定的微管和游离微管蛋白

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

我们的新方案有助于区分和量化管环的三种状态，例如动物组织中的稳定微管、不稳定微管和游离小管。该技术由简单的步骤组成，可以评估管材的结构稳定性，这不能通过对管材平移后修饰的量化来检测。该方法对于研究和开发对微管稳定性至关重要的疾病（如阿尔茨海默病和癌症）的治疗方法至关重要。通过准备用于组织解剖的实验室服装来开始该过程。用碎冰装满一个盒子，然后在上面放两个培养皿。用冰冷的磷酸盐缓冲溶液或PBS填充一个培养皿，用于解剖组织的瞬时洗涤和储存。将用PBS润湿的滤纸放在第二个培养皿上。接下来，称量装有PBS的1.5毫升微管，用于解剖组织储存。从安乐死的小鼠中提取组织后，将它们转移到试管中并重新称量每个微管。将组织移入具有冰冷微管稳定缓冲液或MSB +培养基的玻璃匀浆器中，并立即使用冷冻匀浆器匀浆组织。现在使用巴斯德移液管将组织匀浆转移到两毫升微管中，并在2摄氏度下以2,400G离心三分钟。将上清液转移到新的 1.5 毫升微管中以除去碎屑。在新的 1.5 毫升微管中涡旋上清液或 S1 级分。立即将 200 微升 S1 馏分移液到离心微管中，并使用 TLA-55 转子在 2 摄氏度下以 100， 000 G 离心 20 分钟。第二次离心后，将S2上清液与P2沉淀物分离。立即将全部的 S2 馏分转移到离心微管中，并使用 TLA-120.2 转子在 2 摄氏度下以 500， 000 G 旋转 60 分钟。第三次离心后，将S3上清液与P3沉淀物分离。将整个 S3 馏分溶解在 200 微升 2X 十二烷基硫酸钠或 SDS 样品缓冲液中。将剩余的 S1 级分与等体积的 2X SDS 样品缓冲液混合，用作蛋白质印迹的标准曲线。接下来，向 P2 和 P3 馏分管中加入 400 微升 SDS 样品缓冲液。然后，在将样品转移到新的 1.5 毫升微管中并将它们放入冰箱中之前，短暂地超声处理溶液以溶解沉淀物。将所有样品在 100 摄氏度下煮沸三分钟。P2组分的微管占小鼠脑中α-微管蛋白总量的34.86%，而P3和S3组分的微管分别占56.13%和9.01%。P2、P3和S3组分中β-3微管蛋白的百分比与α-微管蛋白的百分比无显著差异。S2 组分显示微管蛋白通过 300 千道尔顿超滤离心柱的通过有限，而 S3 组分允许微管蛋白复合物完全通过。色谱分离导致 S3 微管蛋白洗脱 100 千道尔顿。在每个馏分中回收等比例的α-和β-微管蛋白。P2组分显著富集乙酰化α-微管蛋白，而酪氨酸化α-微管蛋白在P3组分中占主导地位。在匀浆之前冷冻大脑导致 P2 组分中 α-微管蛋白浓度降低。然而，α-微管蛋白在P3组分中增加。冷冻还降低了 P2 组分中 α-微管蛋白的乙酰化水平并增加了酪氨酸化水平。诺考达唑治疗降低了 P2 组分中的 α-微管蛋白。与冷冻相比，诺考达唑不影响P2微管蛋白翻译后修饰。脑组织中P2微管蛋白浓度显著升高，而P3微管蛋白集中在增殖组织中。Western blotting显示，在神经系统中特异性发现的P2微管蛋白起源于稳定的微管，而P3微管蛋白起源于不稳定的微管。微管的稳定性是高度动态的。在低温环境中快速、不间断地完成该协议非常重要。还要确保组织和馏分在溶解在SDS样品缓冲液中之前没有被冷冻。将使用每个组分的免疫呈递与蛋白质组学相结合，有望确定参与微管动力学的因素。使用这种方法，我们揭示了正常tau蛋白如何存在于微管上。它还可用于研究它在老年大脑中如何变得异常，以确定痴呆的新药物靶点。

Research

JoVE Journal

Biochemistry

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

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

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

科研

生物化学

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Self-Assembly of Microtubule Tactoids

微管触觉的自组装

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

剥离印迹技术：一种冷冻电镜样品制备方法，用于从多层或浓缩生物样品中分离单层

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...