Name: nanogold labeling of the yeast endosomal system for ultrastructural analyses
Uploaded: 2014-07-14T15:00:00
Description: Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

作者感谢勒Scriwanek与图编制的援助。 FR是由ECHO（700.59.003）的支持，ALW打开程序（821.02.017和822.02.014），DFG-NWO合作（DN82-303）和ZonMW VICI（016.130.606）资助。

1，原生质球的制备<ol><li>过夜孵育的酵母在30℃下在10毫升的实验设计来确定适当的介质中。 </li><li>一天后，用光度计测定培养物的光密度在600nm（OD 600），在相同的培养基稀释细胞至OD 600为0.2-0.4，并将它们生长至指数生长期除非的设计实验需要不同的条件。细胞处于指数生长期时，培养具有OD 600为1-2。 </li><li>收集离心10 OD 600细胞等价物，在3,500 xg离心5分钟，?...

<ol>
	<li>Gruenberg, 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+endocytic+pathway:+a+mosaic+of+domains.">The endocytic pathway: a mosaic of domains.</a> Nat Rev Mol Cell Biol. 2, 721-730 (2001).</li><li>Sachse, M., Ramm, G., Strous, G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomes:+multipurpose+designs+for+integrating+housekeeping+and+specialized+tasks.">Endosomes: multipurpose designs for integrating housekeeping and specialized tasks.</a> Histochem Cell Biol. 117, 91-104 (2002).</li><li>Luzio, J. P., Pryor, P. R., Bright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomes:+fusion+and+function.">Lysosomes: fusion and function.</a> Nature Rev Mol Cell Biol. 8, 622-632 (2007).</li><li>Meel, E., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+imagination:+understanding+the+endo-lysosomal+system.">Imaging and imagination: understanding the endo-lysosomal system.</a> Histochem Cell Biol. 129, 253-266 (2008).</li><li>Bonangelino, C. J., Chavez, E. M., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+screen+for+vacuolar+protein+sorting+genes+in+Saccharomyces+cerevisiae.">Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae.</a> Mol Biol Cell. 13, 2486-2501 (2002).</li><li>Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+nanogold-labeled+endocytic+compartments+of+yeast+Saccharomyces+cerevisiae+using+a+cryosectioning+procedure.">Ultrastructural analysis of nanogold-labeled endocytic compartments of yeast Saccharomyces cerevisiae using a cryosectioning procedure.</a> J Histochem Cytochem. 57, 801-809 (2009).</li><li>Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESCRT-III:+an+endosome-associated+heterooligomeric+protein+complex+required+for+MVB+sorting.">ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting.</a> Dev Cell. 3, 271-282 (2002).</li><li>Griffith, J., Mari, M., De Maziere, A., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryosectioning+procedure+for+the+ultrastructural+analysis+and+the+immunogold+labelling+of+yeast+Saccharomyces+cerevisiae.">A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae.</a> Traffic. 9, 1060-1072 (2008).</li><li>Lewis, M. J., Nichols, B. J., Prescianotto-Baschong, C., Riezman, H., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+retrieval+of+the+exocytic+SNARE+Snc1p+from+early+yeast+endosomes.">Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes.</a> Mol Biol Cell. 11, 23-38 (2000).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordering+of+compartments+in+the+yeast+endocytic+pathway.">Ordering of compartments in the yeast endocytic pathway.</a> Traffic. 3, 37-49 (2002).</li><li>Odorizzi, G., Babst, M., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fab1p+PtdIns(3)P+5-kinase+function+essential+for+protein+sorting+in+the+multivesicular+body.">Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body.</a> Cell. 95 (3), 847-858 (1998).</li><li>Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B. M., Slot, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+labeling+of+cryosections+from+high-pressure+frozen+cells.">Immunogold labeling of cryosections from high-pressure frozen cells.</a> Traffic. 8, 471-485 (2007).</li><li>Lah, J. J., Hayes, D. M., Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neutral+pH+silver+development+method+for+the+visualization+of+1-nanometer+gold+particles+in+pre-embedding+electron+microscopic+immunocytochemistry.">A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.</a> J Histochem Cytochem. 38, 503-508 (1990).</li><li>Shah, N., Zhang, S., Harada, S., Smith, R. M., Jarett, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+visualization+of+insulin+translocation+into+the+cytoplasm+and+nuclei+of+intact+H35+hepatoma+cells+using+covalently+linked+Nanogold-insulin.">Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.</a> Endocrinol. 136, 2825-2835 (1995).</li><li>Weipoltshammer, K., Schofer, C., Almeder, M., Wachtler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+enhancement+at+the+electron+microscopic+level+using+Nanogold+and+gold-based+autometallography.">Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.</a> Histochem Cell Biol. 114, 489-495 (2000).</li><li>Tokuyasu, K. 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+technique+for+ultracryotomy+of+cell+suspensions+and+tissues.">A technique for ultracryotomy of cell suspensions and tissues.</a> J Cell Biol. 57, 551-565 (1973).</li><li>Cabrera, 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=Phosphorylation+of+a+membrane+curvature-sensing+motif+switches+function+of+the+HOPS+subunit+Vps41+in+membrane+tethering.">Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering.</a> J Cell Biol. 191, 845-859 (2010).</li><li>Harner, 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=The+mitochondrial+contact+site+complex,+a+determinant+of+mitochondrial+architecture.">The mitochondrial contact site complex, a determinant of mitochondrial architecture.</a> EMBO J. 30, 4356-4370 (2011).</li><li>Nair, U., 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=SNARE+proteins+are+required+for+macroautophagy.">SNARE proteins are required for macroautophagy.</a> Cell. 146, 290-302 (2011).</li><li>Jacquier, 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=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J Cell Sci. 124, 2424-2437 (2011).</li><li>Karanasios, E., 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=Regulation+of+lipid+droplet+and+membrane+biogenesis+by+the+acidic+tail+of+the+phosphatidate+phosphatase+Pah1p.">Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.</a> Mol Biol Cell. 24, 2124-2133 (2013).</li><li>Mari, 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=An+Atg9-containing+compartment+that+functions+in+the+early+steps+of+autophagosome+biogenesis.">An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.</a> J Cell Biol. 190, 1005-1022 (2010).</li><li>Nair, U., 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+role+for+Atg8-PE+deconjugation+in+autophagosome+biogenesis.">A role for Atg8-PE deconjugation in autophagosome biogenesis.</a> Autophagy. 8, 780-793 (2012).</li><li>Vaart, A., Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exit+from+the+Golgi+is+required+for+the+expansion+of+the+autophagosomal+phagophore+in+yeast+Saccharomyces+cerevisiae.">Exit from the Golgi is required for the expansion of the autophagosomal phagophore in yeast Saccharomyces cerevisiae.</a> Mol Biol Cell. 21, 2270-2284 (2010).</li><li>Erpapazoglou, Z., 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+dual+role+for+K63-linked+ubiquitin+chains+in+multivesicular+body+biogenesis+and+cargo+sorting.">A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting.</a> Mol Biol Cell. 23, 2170-2183 (2012).</li><li>Tuinen, E., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+glyceraldehyde-3-phosphate+dehydrogenase,+hexokinase,+and+carboxypeptidase+Y+in+yeast+cells+at+the+ultrastructural+level.">Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.</a> J Histochem Cytochem. 35, 327-333 (1987).</li><li>Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+and+immunolabeling.">Cryosectioning and immunolabeling.</a> Nat Protoc. 2, 2480-2491 (2007).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+the+yeast+endocytic+pathway.">Morphology of the yeast endocytic pathway.</a> Mol Biol Cell. 9, 173-189 (1998).</li><li>Vida, T. A., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vital+stain+for+visualizing+vacuolar+membrane+dynamics+and+endocytosis+in+yeast.">A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.</a> J Cell Biol. 128, 779-792 (1995).</li><li>Hainfeld, J. F., Powell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+frontiers+in+gold+labeling.">New frontiers in gold labeling.</a> J Histochem Cytochem. 48, 471-480 (2000).</li></ol>

免疫电子显微术是一种技术，它允许结合蛋白的定位与在这些蛋白质所在的载体和细胞器的超微结构分辨率。研究了酵母核内体系统时，因为它的车厢出现点状结构，在荧光显微镜中，这一点尤其重要。因此它是难以区分它们。由于这个原因，用一个探针通过EM检测并输入的内吞途径中一个时间依赖的方式是非常重要的特异性标记不同内涵体。这种类型的探针是有价值的，以评估细胞内吞作用...

内体系统中起着至关重要的多种细胞的作用包括贩运溶酶体酶分选受体和质膜（PM）的回收受体1,2的主要膜分拣设备。内涵体被划分在三个不同的车厢， 即 。在早期内涵体（EE），晚期内涵体（LE）和回收内涵体。这种分类是基于它需要的内吞物质到达他们，对特定的标记蛋白，并且在其形态的时间。膜， 即蛋白质和脂质双层，从PM内在设计可以通过内体输送到溶酶体中降解或再循环回。膜也被输送到内涵体从高尔基体，同样，要么继续溶酶体或检索返回到高尔基体。此外，蛋白质可以分为管腔囊泡从内体界膜，这一过程导致形成向内出芽子类别LE时，多泡体。 酵母胞内体系统是相对较不复杂的较高的真核细胞中的一个。酵母胞内体分为EE和LE。相反，哺乳动物细胞中不含有回收内涵体，而且组织特异性溶酶体相关细胞器。因此，他们有一个不太复杂的内体的贩运路线3,4的网络。因此，酵母曾代表仍然表示一个有利的实验系统，研究一些基础原则膜交通的内体系统。这个优点是通过以下事实，涉及该内体途径的许多基因已被初步分离与遗传筛选在酵母5强调。 而酵母胞内体系统中的野生型和突变的细胞已被广泛采用的生化和荧光显微镜方法的研究，其在超微结构水平调查至今只有最小的。形态分析是特别相关的酵母，因为大部分核内体的细胞器被检测为通过荧光显微镜圈点结构，这使得他们难以明确识别6。不幸的是抗血清仅有限数量识别酵母胞内蛋白标志物是工作在免疫电子显微镜（IEM）制剂7-10。对于一些蛋白质，这个问题已经绕过由感兴趣的基因的内源性标记和使用的抗体识别，检测它7,11,12和标签。然而，通常蛋白是因为它们的低表达水平的检测不到的IEM。他们的表达是不是一个解决方案，因为这种方法可以诱导误本地化和/或改建中的细胞器形态/功能。因此，内吞区室中与探针可检测通过电子显微镜EM标签是一种有效的选择。这是特别是如果一个最优解的公关OBE是进入细胞内吞途径中以时间依赖性方式，它允许知道什么时候会标记一个特定的细胞器6。 带正电荷的纳米级金的酵母原生质球（ 即 ，酵母，其中细胞壁已酶促去除）的摄取已经被成功地用于识别酵母胞内隔室10。这些颗粒牢固结合到带负电荷的脂质构成的生物膜。因此，带正电荷的纳米级金的联系人与PM，穿透细胞通过内吞作用和通过EE和LE到达液泡之前。这些小的金颗粒，但是，没有一个合适的尺寸由EM待观察。以使它们可见，它们的大小可通过化学反应，导致银或金的周围的黄金探测器13-15的沉积被放大。我们已经开发并成功应用的IEM方法的基础上，得安方法来执行的亚细胞定位研究8,16。这种方法允许进行酵母制剂免疫标记与形态8,17-24一个出色的分辨率。我们还建立了一个程序结合本IEM协议与酵母核内体系统的纳米金标记舱6。使用这种方法，我们已形态学特征内涵体的不同亚类和超微结构研究突变体与内体运输缺陷6,25。此外，我们已经证明，这种纳米金标记可以与免疫标号提供探索对不同亚群内体目标蛋白的分布的可能性相结合。在这里，我们介绍如何在酵母核内体系统的带正电的纳米金标记实际上是执行。

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">PIPES (piperazin-1,4-bis (2-ethansulfensaure)</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1,102,200,250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">HEPES</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">391,340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">EGTA</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">E4378</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:280px;">MgCl2.6H2O</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1,058,330,250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">DL-Dithiothreitol</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sorbitol</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">S1876</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Positively charged Nanogold</td>
			<td style="width:188px;">Nanoprobes, Yaphank, NY</td>
			<td style="width:119px;">2022</td>
			<td style="width:167px;">store at -20 &#778;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">HQ-silver&#8482; enhancement kit</td>
			<td style="width:188px;">Nanoprobes, Yaphank, NY</td>
			<td style="width:119px;">2012</td>
			<td style="width:167px;">store at -20 &#778;C</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:280px;">Paraformaldehyde (PFA)</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">441244</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:280px;">Glutaraldehyde 8% EM grade</td>
			<td style="width:188px;">Polyscience, Inc. , Warrington, PA</td>
			<td style="width:119px;">216</td>
			<td style="width:167px;">store at 4 &#778;C</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:280px;">Lytic enzyme</td>
			<td style="width:188px;">MP Biomedicals, Santa Ana, CA</td>
			<td style="width:119px;">153526</td>
			<td style="width:167px;">store at 4 &#778;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:280px;">Parafilm M</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">P7793</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 meshes nickel grids&#160;</td>
			<td style="width:188px;">Agar Scientific, Stansted, United Kingdom</td>
			<td style="width:119px;">G209N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;">Cryo-immuno diamond knife 35 &#186;</td>
			<td style="width:188px;">Diatome AG, Biel, Switzerland</td>
			<td style="width:119px;">N.A.</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">UCT ultramicrotome&#160;</td>
			<td style="width:188px;">Leica, Solms, Germany</td>
			<td style="width:119px;">N.A.</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;">Formvar 15/95 resine</td>
			<td style="width:188px;">Electron Microscopy Sciences, Hatfield, PA</td>
			<td style="width:119px;">15800</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 meshes nickel grids&#160;</td>
			<td style="width:188px;">Agar Scientific, Stansted, United Kingdom</td>
			<td style="width:119px;">AGG2050N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Parafilm</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">P7793</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Grids storage box</td>
			<td style="width:188px;">Leica, Solms, Germany</td>
			<td style="width:119px;">162-50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Falcon 100mmx15mm petri dishe&#160;</td>
			<td style="width:188px;">Corning, Corning, NY</td>
			<td style="width:119px;">351029</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pasteur capillary pipettes 150 mm</td>
			<td style="width:188px;">Van Bruggen Glas, Rotterdam, The Netherlands</td>
			<td style="width:119px;">10216234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">1.5 ml microcentrifuge&#160;</td>
			<td style="width:188px;">Sarstedt, N&#252;mbrecht, Germany</td>
			<td style="width:119px;">72690001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 ml tube</td>
			<td style="width:188px;">Bio-one, Alphen aan den Rijn, The Netherlands</td>
			<td style="width:119px;">210296</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Benchkote surface protector</td>
			<td style="width:188px;">Whatman, Maidstone, United Kingdom&#160;</td>
			<td style="width:119px;">1159201</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

nanogold labeling of the yeast endosomal system for ultrastructural analyses

内涵体是主要的膜排序在真核细胞中的检查点中的一个和它们调节回收的蛋白质或毁坏大多从质膜和高尔基体。其结果是内体系统在维持细胞稳态中发挥中心作用，并在属于该网络的细胞器通过小泡运输相互连接的基因的突变，引起严重的病症，包括癌症和神经生物学障碍。因此，黄金的相关性是理解内涵体系统的生物合成和组织相关的机制。 酿酒酵母中一直举足轻重的完成这项任务。要明确标示，并在超微结构水平这一​​模式生物的内体系统的分析，我们在座的带正电荷的纳米金摄取原生质其次是这些粒子通过银增强反应可视化的详细协议。这种方法也是一种宝贵的太l适用于与内体运输缺陷突变体的形态学检查。此外，它不仅适用于超微结构检查，但它也可与免疫标号用于蛋白质定位的调查相结合。

根据所提出的协议，酵母核内体系统的形态可以是由接入传输的EM。 图1显示了不同类型的内体区室中已达成，因此，标记的纳米金。银增强纳米金可以清楚地看到作为电子致密颗粒。建立了银增强反应的最佳设置允许具有在5至15纳米，不与酵母细胞的超微结构的干扰之间的均匀粒度范围的金颗粒。质膜以及早期内涵体车厢都可以访问的纳米金5分钟孵化在24°C（ 图1A）后?...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

酵母核内体系统的超微结构分析的纳米金标记

酵母， 酿酒酵母 ，一直是一个关键模式生物识别和研究基因调节内体系统的生物合成和功能。在这里，我们提出了内体车厢的超微结构研究特定的标签一个详细的协议。

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Research

JoVE Journal

Biology

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

被截肢者的智能植入种植体设计系统的生物电分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

Fluorescent Labeling of Drosophila Heart Structures

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...