Name: isolation and purification of kinesin from drosophila embryos
Uploaded: 2012-04-27T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Purification of Kinesin from Drosophila Embryos at JoVE.com

特别感谢克里斯艺和贾森德尔里奥在这个项目的支持和援助。这项工作被授予GM070676石药RO1的支持。

苍蝇可以购买，并在实验室中扩增菌株。根据对果蝇文化收到小瓶量，需要频繁地“翻转”文化小瓶，小瓶已达到最大容量。扩增继续填充，直到大约50 果蝇的培养瓶。然后，一个“飞杯”三角帽100毫升烧杯（飞杯决策将在下一节讨论）。 24小时后，使用胚胎从这些杯种子新果 ​​蝇的培养瓶，贴在底部飞食品。在室温下从隔夜胚胎全面增长的苍蝇， 果蝇胚胎的生长?...

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	<li>Ashburner, M., Thompson, J. N., Ashburner, M., Wright, T. R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+culture+of+Drosophila+2A.">The laboratory culture of Drosophila 2A.</a> The genetics and biology of Drosophila. , 1-81 (1978).</li><li>Roberts, D. 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> Drosophila: A Practical Approach. , (1998).</li><li>Kunert, N., Brehm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+Drosophila+Embryosand+Chromatographic+Purification+of+Native+Protein+Complexes.">Mass production of Drosophila Embryosand Chromatographic Purification of Native Protein Complexes.</a> Methods in Molecular Biology. 420, 359 (2008).</li><li>Goldstein, L. S. B., Fyrberg, E. A., Wilson, L., Matsudaira, P. T., Eds, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+Practical+Uses+in+Cell+and+Molecular+Biology.">Drosophila melanogaster: Practical Uses in Cell and Molecular Biology.</a> Methods in Cell Biology. 44, (1994).</li><li>Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+kinesin:+Characterization+of+microtubule+motility+and+ATPase.">Drosophila kinesin: Characterization of microtubule motility and ATPase.</a> Proceedings of the National Academy of Science. 85, 1109 (1988).</li><li>Shubeita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1098 (2008).</li><li>Svoboda, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+Velocity+Measured+for+Single+Kinesin+Molecules.">Force and Velocity Measured for Single Kinesin Molecules.</a> Cell. 77, 773 (1994).</li><li>Cole, D. G., Saxton, W. M., Sheehan, K. B., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"Slow"+Homotetrameric+Kinesin-related+Motor+Protein+Purified+from+Drosophila+embryos.">A "Slow" Homotetrameric Kinesin-related Motor Protein Purified from Drosophila embryos.</a> J. Biol. Chem. 269, 22917-22917 (1994).</li><li>Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Analysis+of+Microtuble+Motor+Proteins.">Isolation and Analysis of Microtuble Motor Proteins.</a> Drosophila Melanogaster. Bloomington: Academic. 44, 279 (1994).</li><li>Scholey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+Assay+for+Motor+Proteins.">Motility Assay for Motor Proteins.</a> Methods in Cell Biology. 39, 138 (1993).</li><li>Wagner, M. C., Pfister, K., Brody, S., Bloom, G. <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+kinesin+from+bovine+brain+and+assay+of+microtubule-stimulated+ATPase+activity.">Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity.</a> Methods in Enzymology. 196, 157 (1991).</li><li>Aizawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+Family+in+Murine+Nerwous+System.">Kinesin Family in Murine Nerwous System.</a> The Journal of Cell Biology. 119, 1287-1287 (1992).</li><li>Ori-McKenney, K. M., Xu, J., Gross, S. P., 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+cytoplasmic+dynein+tail+mutation+impairs+motor+processivity.">A cytoplasmic dynein tail mutation impairs motor processivity.</a> Nature Cell Biology. 12, 1228-1228 (2010).</li></ol>

牛脑是最广泛使用的起始原料11净化全长kinesin的，虽然小鼠的大脑已被使用，以及12。使用牛脑动蛋白源作为一个大的缺点是新鲜的起始原料的可用性：屠宰场通常无法进入，必须非常新鲜，以取得积极的kinesin的大脑。此外，青年奶牛的大脑是有效的。最后，牛的基因操纵，目前没有一个可行的选择，所以只有野生型的蛋白质进行研究。 小鼠更容易，但收获?...

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isolation and purification of kinesin from drosophila embryos

马达蛋白沿着微管移动的货物，并运送到特定的亚细胞位置。由于改变交通建议多种神经退行性疾病的基础，了解微管的汽车运输及其调控最终可能会导致更好的治疗方法。动蛋白是真核细胞马达蛋白在ATP水解供电，沿微管（MTS）顺（加末）的方向移动。在这里，我们报告了详细的净化从果蝇胚胎中分离的积极全长kinesin的协议，从而使单分子生物物理研究果蝇遗传学相结合。拥有大约1000名女性，每杯，约50铺设杯开始，我们进行了隔夜的集合。这提供了约10毫升装的胚胎。胚胎漂白dechorionated（产生的胚胎约9克），然后均匀。 AF之三的破坏，匀浆，利用低速旋转，由高速离心澄清。澄清的上清液治疗GTP和紫杉醇微管聚合。动蛋白聚合微管上，通过增加ATP的模拟在室温下，5&#39;-腺苷酸imidodiphosphate固定。 kinesin的绑定后，微管沉降的通过通过蔗糖坐垫高速离心。微球，然后再暂停，并重复此过程。最后，ATP的释放从MTS动蛋白。高速离心，然后降速MTS，留在上清动蛋白。这kinesin的使用100第纳尔切断进一步净化过滤离心过滤，分装，捕捉液氮冷冻，储存在-80°C。使用纯化的样品进行SDS凝胶电泳和免疫印迹。最后离心过滤步骤之前和之后的纯化的样品电机活动在体外单分子微管法进行了评估。离心过滤前后动蛋白组分显示processivity以前文献报道。进一步的实验正在进行评估动蛋白和其他运输相关的蛋白质之间的相互作用。

Watch this Scientific Journal Video about Isolation and Purification of Kinesin from Drosophila Embryos at JoVE.com

Isolation and Purification of Kinesin from Drosophila Embryos

这是一个协议隔离积极全长驱动蛋白<em&gt;果蝇</em&gt;单分子生物物理研究的胚胎。我们如何收集胚胎，使胚胎裂解，然后聚合微管（MTS）。动蛋白纯化固定在MTS，纺纱动蛋白吨复合物，然后释放从MTS动蛋白通过磷酸腺苷此外。

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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Biology

Isolation and Purification of Kinesin from Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

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Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

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无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

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直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

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原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Purification of Transcripts and Metabolites from Drosophila Heads

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成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

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In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

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一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...