Name: visualization of minisog tagged dna repair proteins in combination with electron spectroscopic imaging (esi)
Uploaded: 2015-09-24T14:00:00
Description: Watch this Scientific Journal Video about Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI at JoVE.com

We thank Dr. Roger Tsien for providing us with the miniSOG constructs. Dr. Xuejun Sun for help with the TEM. Lisa Lem, and Peter Shipple from the Cross Cancer Institute for supplying oxygen. Hilmar Strickfaden holds a postdoctoral fellowship by the Alberta Cancer foundation and was supported by the Bayrische Forschungsallianz. This work was supported by grants from the Canadian Institutes of Health Research and Alberta Cancer Foundation.

1.代miniSOG细胞系<ol><li>生长的U2OS（人骨肉瘤）细胞在含2ml即补充有10％胎牛血清（FBS），在37℃下在湿润的培养箱中5％的CO 2低葡萄糖Dulbecco改良的Eagle培养基（DMEM）的35mm培养皿气氛。 </li><li>转染的细胞时，它们是80％汇合通过脂质转染法，用纯化的转染质量（A269 / 280比率1.8-2.0）包含miniSOG和mCherry序列质粒构建根据转染试剂19的制造商的协议标签修复蛋白。该miniSOG表达质粒可以?...

<ol>
	<li>Schermelleh, L., Heintzmann, R., Leonhardt, 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+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of Cell Biology. 190, 165-175 (2010).</li><li>Bazett-Jones, D. P., Hendzel, M. J., Kruhlak, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometric+analysis+of+protein-+and+nucleic+acid-based+structures+in+the+cell+nucleus.">Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus.</a> Micron (Oxford, England: 1993). 30, 151-157 (1999).</li><li>Michael J Hendzel, F. M. B., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Visualization+of+a+Protein+Nuclear+Architecture.">Direct Visualization of a Protein Nuclear Architecture.</a> Molecular biology of the cell. 10, 2051 (1999).</li><li>Ottensmeyer, F. P., Andrew, 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=High-resolution+microanalysis+of+biological+specimens+by+electron+energy+loss+spectroscopy+and+by+electron+spectroscopic+imaging.">High-resolution microanalysis of biological specimens by electron energy loss spectroscopy and by electron spectroscopic imaging.</a> Journal of ultrastructure research. 72, 336-348 (1980).</li><li>Leapman, R. D., Ornberg, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+electron+energy+loss+spectroscopy+in+biology.">Quantitative electron energy loss spectroscopy in biology.</a> Ultramicroscopy. 24, 251-268 (1988).</li><li>Simon, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spectroscopic+imaging.">Electron spectroscopic imaging.</a> Ultrastructural pathology. 11, 705-710 (1987).</li><li>Egerton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Energy-Loss Spectroscopy in the Electron Microscope. , (2011).</li><li>Aronova, M. A., Kim, Y. C., Zhang, G., Leapman, 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=Quantification+and+thickness+correction+of+EFTEM+phosphorus+maps.">Quantification and thickness correction of EFTEM phosphorus maps.</a> Ultramicroscopy. 107, 232-244 (2007).</li><li>Hopkins, C., Gibson, A., Stinchcombe, J., Futter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+molecules+employing+horseradish+peroxidase+as+reporter+enzyme+for+protein+localization+in+the+electron+microscope.">Chimeric molecules employing horseradish peroxidase as reporter enzyme for protein localization in the electron microscope.</a> Methods in enzymology. 327, 35-45 (2000).</li><li>Shu, X., 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+Genetically+Encoded+Tag+for+Correlated+Light+and+Electron+Microscopy+of+Intact+Cells,+Tissues,+and+Organisms.">A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms.</a> PLoS biology. 9, e1001041 (2011).</li><li>Gaietta, G., 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=Multicolor+and+electron+microscopic+imaging+of+connexin+trafficking.">Multicolor and electron microscopic imaging of connexin trafficking.</a> Science (New York, NY). 296, 503-507 (2002).</li><li>Fernandez-Capetillo, O., Celeste, A., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+on+foci:+H2AX+and+the+recruitment+of+DNA-damage+response+factors.">Focusing on foci: H2AX and the recruitment of DNA-damage response factors.</a> Cell cycle (Georgetown, Tex). 2, 426-427 (2003).</li><li>Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+foci+of+mammalian+Rad51+recombination+protein+in+somatic+cells+after+DNA+damage+and+its+localization+in+synaptonemal+complexes.">Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 2298-2302 (1995).</li><li>Maser, R. S., Monsen, K. J., Nelms, B. E., Petrini, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hMre11+and+hRad50+nuclear+foci+are+induced+during+the+normal+cellular+response+to+DNA+double-strand+breaks.">hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks.</a> Molecular and Cellular Biology. 17, 6087-6096 (1997).</li><li>Bekker-Jensen, S., Mailand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+function+of+DNA+double-strand+break+repair+foci+in+mammalian+cells.">Assembly and function of DNA double-strand break repair foci in mammalian cells.</a> DNA repair. 9, 1219-1228 (2010).</li><li>Chapman, J. R., Sossick, A. J., Boulton, S. J., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1-associated+exclusion+of+53BP1+from+DNA+damage+sites+underlies+temporal+control+of+DNA+repair.">BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair.</a> Journal of cell science. 125, 3529-3534 (2012).</li><li>Dellaire, G., Kepkay, R., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+imaging+of+changes+in+the+structure+and+spatial+organization+of+chromatin,+gamma-H2A.X+and+the+MRN+complex+within+etoposide-induced+DNA+repair+foci.">High resolution imaging of changes in the structure and spatial organization of chromatin, gamma-H2A.X and the MRN complex within etoposide-induced DNA repair foci.</a> Cell cycle (Georgetown, Tex). 8, 3750-3769 (2009).</li><li>Kruhlak, M. 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=Changes+in+chromatin+structure+and+mobility+in+living+cells+at+sites+of+DNA+double-strand+breaks.">Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.</a> The Journal of Cell Biology. 172, 823-834 (2006).</li><li>Goodson, H. V., Dzurisin, J. S., Wadsworth, P. <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+stable+cell+lines+expressing+GFP-tubulin+and+photoactivatable-GFP-tubulin+and+characterization+of+clones.">Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP-tubulin and characterization of clones.</a> Cold Spring Harbor protocols. 2010, (2010).</li><li>Zeyda, M., Borth, N., Kunert, R., Katinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+sorting+conditions+for+the+selection+of+stable,+high-producing+mammalian+cell+lines.">Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines.</a> Biotechnology progress. 15, 953-957 (1999).</li><li>Dellaire, G., Nisman, R., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light+and+electron+spectroscopic+imaging+of+chromatin+in+situ.">Correlative light and electron spectroscopic imaging of chromatin in situ.</a> Methods in enzymology. , 375-456 (2004).</li><li>Campbell, S., Ismail, I. H., Young, L. C., Poirier, G. G., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+repressive+complex+2+contributes+to+DNA+double-strand+break+repair.">Polycomb repressive complex 2 contributes to DNA double-strand break repair.</a> Cell cycle. 12, 2675-2683 (2013).</li><li>Mortusewicz, 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=Recruitment+of+RNA+polymerase+II+cofactor+PC4+to+DNA+damage+sites.">Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites.</a> J Cell Biol. 183, 769-776 (2008).</li><li>Bekker-Jensen, 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=Spatial+organization+of+the+mammalian+genome+surveillance+machinery+in+response+to+DNA+strand+breaks.">Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.</a> The Journal of Cell Biology. 173, 195-206 (2006).</li><li>Ferrando-May, 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=Highlighting+the+DNA+damage+response+with+ultrashort+laser+pulses+in+the+near+infrared+and+kinetic+modeling.">Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.</a> Frontiers in genetics. 4, 135 (2013).</li><li>Lukas, 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=53BP1+nuclear+bodies+form+around+DNA+lesions+generated+by+mitotic+transmission+of+chromosomes+under+replication+stress.">53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress.</a> Nature. 13, 243-253 (2011).</li><li>Harrigan, J. 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=Replication+stress+induces+53BP1-containing+OPT+domains+in+G1+cells.">Replication stress induces 53BP1-containing OPT domains in G1 cells.</a> The Journal of Cell Biology. 193, 97-108 (2011).</li><li>Aronova, M. 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=Reprint+of+'Three-dimensional+elemental+mapping+of+phosphorus+by+quantitative+electron+spectroscopic+tomography+(QuEST).">Reprint of 'Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST).</a> J. Struct. Biol. 161, 322-335 (2007).</li><li>Fussner, 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=Constitutive+heterochromatin+reorganization+during+somatic+cell+reprogramming.">Constitutive heterochromatin reorganization during somatic cell reprogramming.</a> The EMBO journal. 30, 1778-1789 (2011).</li><li>Kepkay, R., Attwood, K. M., Ziv, Y., Shiloh, Y., Dellaire, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KAP1+depletion+increases+PML+nuclear+body+number+in+concert+with+ultrastructural+changes+in+chromatin.">KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin.</a> Cell cycle. 10, 308-322 (2011).</li></ol>

ESI可作为极好的工具用于检查染色质和核糖核蛋白的不同状态在细胞核中，因为它是能够专门映射是富含磷的区域。它深刻增强细节，可以用电子显微镜来获得并且不依赖于非特异性对比方法的量。 ESI的一个缺点是，它需要比在相同的加速电压常规的TEM较薄部分。这可以通过在与连续切片或使用层析方法28来克服。 虽然大量的信息可以通过查看磷和氮的分布来获得?...

尽管近年来在1光学显微镜显著的进步，细胞生物学家仍在遭受分辨率的差距。这限制涉及大分子复合物之间的协调相互作用中基本的细胞过程中的结构-功能关系的了解（例如，在染色质重塑，DNA修复，转录的RNA和DNA复制）。虽然透射电子显微镜（TEM）■提供所要求的分辨率，已经挑战来定义这些过程无法的结构因为标注的特定蛋白质，同时还能够以确定的可视化结构的生化成分。在没有内部的膜，以帮助区分核结构中，核一直特别具有挑战性。电子光谱成像（ESI）通过允许DNA，RNA和PROT的同时检测和分化解决某些限制EIN基核结构2-5。 电子光谱成像： 为了映射以高灵敏度和分辨率在电子显微镜下元素分布，可以用一个图像光谱仪，其选择已通过与元素的在样本 6内壳层电子相互作用非弹性散射电子。因为能量的单元特定的量被丢失原子在试样的电离的结果，这些电子可以被分离并使用被附接到电子显微镜的分光计可视化。因此，已经相互作用与试样的电子的频谱分析揭示关于样品7的元素组成的定性和定量的信息。电子通过检体传递时不损失能量被发现在电子能量损失的“零损耗峰”谱。这些电子的丰度是相关的试样的质量，密度和厚度，并包括通过检体通过，而不碰撞与样品或通过试样通道中失去能量的电子。这个信息可以是为特定的元素本的原子数，在试样8的绝对定量是有用的。 因为生物样品包括主要光元件很差偏转电子在TEM的入射光束，染色方法使用重金属盐具有以生成所述样品中的对比度得以应用。特异性大多数这些造影剂和无法的可视化多个污点其中特异性是可能的缺乏在细胞核的研究已限制常规电子显微镜的值。 ESI有超过常规的TEM显著的优点，特别是对细胞核的结构的研究。有可能利用DNA和含有RNA的大分子复合的富磷性质区分蛋白质复合核蛋白复合物和解决基于核酸它们的密度不同核蛋白复合物。剩余的生物材料可以根据它的丰度氮气进行成像。不同的解剖结构中的映射只是这两种元素的分布和相对丰度的分析为我们提供了许多关于核信息。例如，它是容易识别染色质，并在地图上的核糖体代表磷丰度。所述interchromatin空间，核孔复合物，和核机构，另一方面，可以容易地在该氮地图图像中检测到。 迷你单态氧发生器系统（miniSOG） 尽管ESI代表一种强大的技术原位染色质结构来研究，因为它需要的优势在磷和氮之间的元素组成的特征比率，元素组成通常不能使用的蛋白质复合物的不同群体之间进行区分。抗体标记的小的金颗粒在纳米范围内已被广泛地用于绘制单个分子的位置。由于金颗粒通常连接到一个二级抗体，它将出现的周围由第一抗体检测的表位大约20nm的圆周内。在后包埋样品，抗体只能检测是在部分的表面露出的表位。虽然这是可能的，以证明抗原的存在，并将其涉及到细胞的特定解剖结构，所获得的信息是不完整的，因为大部分的表位是由树脂遮蔽。预嵌入技术，其使用类似的协议与用于荧光显微镜，在整个允许访问抗原样品的整个深度，但所需要的使抗体穿透细胞通常需要去除脂膜，并删除无法固定醛组分通透步骤。此外，优选的醛固定液超微结构保存，戊二醛，通常破坏的表位，因此，多聚甲醛，通常需要。这是在蛋白质 - 蛋白质交联效果较差。抗体的被标记有金纳米颗粒的另一缺点是，金是创建一个强烈的对比，可以掩盖样品，它具有弱对比度性的结构细节非常电子致密材料。 绿色荧光蛋白（GFP）作为表达的蛋白质标签的出现改变了使用荧光显微镜的解答在细胞生物学的问题。标记的蛋白质与小荧光域允许其在体内的分布制图</e米&gt;而不需要透过程，可以改变结构。为了翻译用蛋白质标记映射蛋白质分布在细胞中，以电子显微镜的优雅原则，需要一个系统，该系统能够产生一个信号接近感兴趣的结构，并产生以TEM对比度。聚合的二氨基联苯胺]是经常用于组织学检测抗体结合的污点。此染色是使用辣根过氧化物酶（HRP）缀合的第二抗体通常沉积。尽管反应产生一个可靠的结果，HRP是不是在细胞9的胞质溶胶催化活性。 HRP的反应产物也可以扩散远离一代的网站，这样它们的分辨率比纳米金法10恶化。为了绕开这些问题，FLASH / ReAsH系统的开发11。它由重组融合蛋白，其拥有四半胱氨酸基序。这个主题可以结合一个biarsenic荧光。当激发时，结合的闪光或ReAsH是能够产生反应性高的单重态氧，因此photoconvert二氨基联苯胺（DAB）插入在标签的蛋白质的位点紧接该沉淀的聚合物。民建联聚合物可染色用四氧化锇，这是电子致密，因而可以用于映射重组融合蛋白在TEM的分布。 在2011年，舒等人 10提交的miniSOG系统，它由来自拟南芥的黄素蛋白是荧光，并且能够在与448纳米的蓝色光激发而产生很多单线态氧自由基衍生的小106个氨基酸融合标记的。这些单线态氧自由基可用于光氧化二氨基联苯胺，以形成上和标签蛋白，这是比免疫标记的抗体11相当接近的表面附近的聚合物。虽然闪存/ ReAsH系统需要使氟铬和二氨基联苯胺成执行光转化前的细胞中，miniSOG系统只需要二氨基联苯胺和轻，此外是大约两倍于聚合二氨基联苯胺作为有效。在这里，miniSOG采用与ESI的组合，以图的DNA修复灶的超微结构。 DNA修复灶（DRF） 未修复DNA双链断裂构成严重威胁到细胞，因为它们可以导致易位和遗传信息的丢失。反过来，这可导致衰老，癌症和细胞死亡。参与DNA双链断裂修复，许多蛋白积聚在病灶周围的DNA双链组装打破12-14。虽然它们的功能是未知的，它们所代表的部位在包含DNA双链断裂，并且是DNA双链断裂修复的部位的核心。 DNA修复FOCI（DRF）的特点是荧光显微镜和它们作为生物标志物用于DNA损伤12,15。它们大量相对于双链断裂的大小和被认为是相对均匀的，直到最近的超高分辨率显微镜研究揭示在每个聚焦16个子条块分子的一些证据。为了了解这些位点的组织，有必要以可视化的所有潜在的生物结构的相对于彼此。这不能用荧光显微镜下进行，但有可能通过电子显微镜17,18。这里，描述了一种方法，结合了电子光谱成像与miniSOG方法，来说明这种组合方法的潜力，以探索的DNA双链断裂修复的超微结构。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>35 mm glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Gridded 35 mm glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-2-14-CGRD&#160;</td>
			<td>not made for immersion objectives</td>
		</tr>
		<tr>
			<td>LR White: acryl resin</td>
			<td>emsdiasum</td>
			<td>14381</td>
			<td></td>
		</tr>
		<tr>
			<td>LR White accelerator</td>
			<td>emsdiasum</td>
			<td>14385</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;-Diaminobenzidine tetrahydrochloride 10 mg tablets</td>
			<td>Sigma</td>
			<td>D5905</td>
			<td></td>
		</tr>
		<tr>
			<td>Slim Bar Grids 300 Mesh</td>
			<td>SPI</td>
			<td>1161123</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten-Point Lab Pen</td>
			<td>emsdiasum</td>
			<td>41148</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide</td>
			<td>emsdiasum</td>
			<td>19100</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Coater 208carbon</td>
			<td>Cressington</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra microtome Leica EM UC6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop CS5</td>
			<td>Adobe</td>
			<td></td>
			<td>might even work with older versions</td>
		</tr>
		<tr>
			<td>Digital Micrograph V.2.30</td>
			<td>Gatan</td>
			<td></td>
			<td>might even work with older versions</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma</td>
			<td>H-1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Effectene</td>
			<td>Quiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniSOG-Constructs</td>
			<td>Tsien-Lab</td>
			<td></td>
			<td><a href="mailto:tsienlab@yahoo.com">tsienlab@yahoo.com</a></td>
		</tr>
		<tr>
			<td>MDC1 miniSOG mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>53BP1 miniSOG mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rad52 miniSOG mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cesium 137 radiation source &#34;MARK 1&#34;</td>
			<td>&#160;(J.L. Shepherd &#38; Associated)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ/FiJi</td>
			<td>open source</td>
			<td></td>
			<td>http://fiji.sc/Fiji</td>
		</tr>
		<tr>
			<td>2 ml Eppendorf tubes</td>
			<td>Fisherbrand</td>
			<td>05-408-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Knife ultra 35&#176;</td>
			<td>Diatome</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming Knife ultratrim</td>
			<td>Diatome</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cacodylate trihydrate</td>
			<td>emsdiasum</td>
			<td>12300</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Glutaraldehyde EM Grade 8%</td>
			<td>emsdiasum</td>
			<td>16020</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>emsdiasum</td>
			<td>21180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>emsdiasum</td>
			<td>21190</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>emsdiasum</td>
			<td>19202</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide 4% solution</td>
			<td>emsdiasum</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Micoscope Axiovert 200M</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisherbrand</td>
			<td>A142-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Solution 10M</td>
			<td>Fluka</td>
			<td>72068</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Medigas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-Amino-1,2,4-triazole</td>
			<td>Sigma</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium cyanide</td>
			<td>Sigma</td>
			<td>207810</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>emsdiasum</td>
			<td>15058</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Razor blade Single Edge Carbon Steel</td>
			<td>emsdiasum</td>
			<td>71960</td>
			<td>Caution Sharp!</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D 5546</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>lifetechnologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>lifetechnologies</td>
			<td>11811-023</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope 200 kV</td>
			<td>JEOL</td>
			<td>2100</td>
			<td></td>
		</tr>
		<tr>
			<td>GIF Tridiem 863 Energy filter</td>
			<td>Gatan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of minisog tagged dna repair proteins in combination with electron spectroscopic imaging (esi)

极限到光学分辨率和鉴定特定蛋白质种群透射电镜的挑战已经在细胞生物学障碍。许多现象无法通过体外分析在简化的系统进行说明，并且需要额外的结构信息在原位 ，特别是在1毫微米和0.1微米之间的范围内，以便被完全理解。这里，电子光谱成像，透射电子显微技术，允许蛋白质和核酸的分布同时映射，和表达标签，miniSOG，被组合以研究DNA双链断裂修复病灶的结构和组织。

ESI 与常规的TEM图像比较所述核（ 图1）的ESI 图像 （例如， 图7-1）中揭示了解剖结构，可以很容易分辨的显着增加。染色质脊显示为黄色，这是很容易识别在小鼠细胞中的chromocenters。核仁可以容易地从染色质区分通过圆形结构和不同的颜色，由于预组装的核糖体已经含有高量的蛋白质，其是富含氮，除了核糖核酸，其中富?...

Watch this Scientific Journal Video about Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI at JoVE.com

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Research

JoVE Journal

Biology

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...