Name: isolation of physiologically active thylakoids and their use in energy-dependent protein transport assays
Uploaded: 2018-09-28T14:45:00
Description: Watch this Scientific Journal Video about Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays at JoVE.com

这份手稿是由化学科学、地球科学和生物科学司资助的, 408 美国能源部基础能源科学办公室通过赠款 DE-SC0017035

1. 初始材料
<ol><li>在400毫升蒸馏水中浸泡大约55克豌豆3小时, 然后在一个塑料托盘 (35 厘米 x 20 厘米 x 6 厘米) 在覆盖着薄薄的蛭石层的土壤中播种。</li>
	<li>将豌豆的托盘生长在20摄氏度以下12/12 小时/暗 (50 µE/米2s) 周期9至15天。</li>
	<li>根据首选方法制备蛋白质基质。 注: 我们已经准备了蛋白质基质使用多种方法, 包括 1) 从纯化质粒体外转录, 然后用小麦胚芽提取物或兔网织裂解...

<ol>
	<li>Ellis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+protein+synthesis:+principles+and+problems.">Chloroplast protein synthesis: principles and problems.</a> Sub-cellular biochemistry. 9, 237 (1983).</li><li>Li, H. -. m., Chiu, 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=Protein+transport+into+chloroplasts.">Protein transport into chloroplasts.</a> Annual review of plant biology. 61, (2010).</li><li>Cline, K., Ettinger, W., Theg, 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=Protein-specific+energy+requirements+for+protein+transport+across+or+into+thylakoid+membranes.+Two+lumenal+proteins+are+transported+in+the+absence+of+ATP.">Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.</a> Journal of Biological Chemistry. 267 (4), 2688-2696 (1992).</li><li>Skalitzky, C. 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=Plastids+contain+a+second+sec+translocase+system+with+essential+functions.">Plastids contain a second sec translocase system with essential functions.</a> Plant physiology. 155 (1), 354-369 (2011).</li><li>Dabney-Smith, C., Storm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastid Biology. , 271-289 (2014).</li><li>Kim, S. J., Jansson, S., Hoffman, N. E., Robinson, C., Mant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+&amp;#34;assisted&amp;#34;+and+&amp;#34;spontaneous&amp;#34;+mechanisms+for+the+insertion+of+polytopic+chlorophyll-binding+proteins+into+the+thylakoid+membrane.">Distinct &amp;#34;assisted&amp;#34; and &amp;#34;spontaneous&amp;#34; mechanisms for the insertion of polytopic chlorophyll-binding proteins into the thylakoid membrane.</a> Journal of Biological Chemistry. 274 (8), 4715-4721 (1999).</li><li>Emanuelsson, O., Nielsen, H., Von Heijne, G. C. h. l. o. r. o. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChloroP,+a+neural+network-based+method+for+predicting+chloroplast+transit+peptides+and+their+cleavage+sites.">ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites.</a> Protein Science. 8 (5), 978-984 (1999).</li><li>Emanuelsson, O., Brunak, S., Von Heijne, G., Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locating+proteins+in+the+cell+using+TargetP,+SignalP+and+related+tools.">Locating proteins in the cell using TargetP, SignalP and related tools.</a> Nature protocols. 2 (4), 953 (2007).</li><li>Ling, Q., Jarvis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+import+into+chloroplasts+isolated+from+stressed+plants.">Analysis of protein import into chloroplasts isolated from stressed plants.</a> Journal of Visualized Experiments. (117), e54717 (2016).</li><li>Lo, S. M., Theg, 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=.">.</a> Photosynthesis Research Protocols. , 139-157 (2011).</li><li>Vernon, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+chlorophylls+and+pheophytins+in+plant+extracts.">Spectrophotometric determination of chlorophylls and pheophytins in plant extracts.</a> Analytical Chemistry. 32 (9), 1144-1150 (1960).</li><li>Knott, T. G., Robinson, 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+secA+inhibitor,+azide,+reversibly+blocks+the+translocation+of+a+subset+of+proteins+across+the+chloroplast+thylakoid+membrane.">The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane.</a> Journal of Biological Chemistry. 269 (11), 7843-7846 (1994).</li><li>Yuan, J., Henry, R., McCaffery, M., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SecA+homolog+in+protein+transport+within+chloroplasts:+evidence+for+endosymbiont-derived+sorting.">SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting.</a> Science. 266 (5186), 796-798 (1994).</li><li>Nohara, T., Nakai, M., Goto, A., Endo, T. <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+characterization+of+the+cDNA+for+pea+chloroplast+SecA+Evolutionary+conservation+of+the+bacterial-type+SecA-dependent+protein+transport+within+chloroplasts.">Isolation and characterization of the cDNA for pea chloroplast SecA Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts.</a> FEBS letters. 364 (3), 305-308 (1995).</li><li>Endow, J. K., Singhal, R., Fernandez, D. E., Inoue, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone-assisted+post-translational+transport+of+plastidic+type+I+signal+peptidase+1.">Chaperone-assisted post-translational transport of plastidic type I signal peptidase 1.</a> Journal of Biological Chemistry. 290 (48), 28778-28791 (2015).</li><li>Luirink, J., Sinning, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRP-mediated+protein+targeting:+structure+and+function+revisited.">SRP-mediated protein targeting: structure and function revisited.</a> Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1694 (1-3), 17-35 (2004).</li><li>Yuan, J., Henry, R., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+factor+plays+an+essential+role+in+protein+integration+into+thylakoids+that+cannot+be+replaced+by+unfolding+or+by+heat+shock+protein.">Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein.</a> Hsp70. Proceedings of the National Academy of Sciences. 90 (18), 8552-8556 (1993).</li><li>Tjalsma, H., van Dijl, 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=Proteomics-based+consensus+prediction+of+protein+retention+in+a+bacterial+membrane.">Proteomics-based consensus prediction of protein retention in a bacterial membrane.</a> Proteomics. 5 (17), 4472-4482 (2005).</li><li>Widdick, D. A., Eijlander, R. T., van Dijl, J. M., Kuipers, O. P., Palmer, 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+Facile+Reporter+System+for+the+Experimental+Identification+of+Twin-Arginine+Translocation+(Tat)+Signal+Peptides+from+All+Kingdoms+of+Life.">A Facile Reporter System for the Experimental Identification of Twin-Arginine Translocation (Tat) Signal Peptides from All Kingdoms of Life.</a> Journal of Molecular Biology. 375 (3), 595-603 (2008).</li><li>Yuan, J., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastocyanin+and+the+33-kDa+subunit+of+the+oxygen-evolving+complex+are+transported+into+thylakoids+with+similar+requirements+as+predicted+from+pathway+specificity.">Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity.</a> Journal of Biological Chemistry. 269 (28), 18463-18467 (1994).</li><li>Kirchhoff, H., Borinski, M., Lenhert, S., Chi, L., B&#252;chel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transversal+and+lateral+exciton+energy+transfer+in+grana+thylakoids+of+spinach.">Transversal and lateral exciton energy transfer in grana thylakoids of spinach.</a> Biochemistry. 43 (45), 14508-14516 (2004).</li><li>Frielingsdorf, S., Jakob, M., Kl&#246;sgen, 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+stromal+pool+of+TatA+promotes+Tat-dependent+protein+transport+across+the+thylakoid+membrane.">A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane.</a> Journal of Biological Chemistry. 283 (49), 33838-33845 (2008).</li><li>Tu, C. -. J., Schuenemann, D., Hoffman, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+FtsY,+chloroplast+signal+recognition+particle,+and+GTP+are+required+to+reconstitute+the+soluble+phase+of+light-harvesting+chlorophyll+protein+transport+into+thylakoid+membranes.">Chloroplast FtsY, chloroplast signal recognition particle, and GTP are required to reconstitute the soluble phase of light-harvesting chlorophyll protein transport into thylakoid membranes.</a> Journal of Biological Chemistry. 274 (38), 27219-27224 (1999).</li></ol>

叶绿体和囊体分离
过度破损会导致叶绿体分离不良, 而在梯度分离后, 不良的囊体产生。最好通过确保所有物质在十五年代的混合和脉动之前被浸没, 直到完全匀质之前, 轻轻地融汇收获的组织。如有必要, 在每回合中使用较少组织的多轮混合。
冷藏所有与收获组织接触的材料可以帮助分离的叶绿体保持2小时的活动。这是重要的是保持在冰上的叶绿体在黑暗?...

几乎所有负责适当叶绿体功能的蛋白质机械都必须从细胞质1移位。在叶绿体信封, 蛋白质基质是通过 translocon 的外层膜 (TOC) 和 translocon 的内膜 (TIC)2进口。进一步靶向囊体膜和流明发生通过双精氨酸易位 (cpTat)3, 分泌 (cpSec1)4, 信号识别粒子 (cpSRP)5和自发插入通路6.一种对生理活性叶绿体和类囊体膜进行高产分离的方法, 是测量易位事件的热力学和动力学的必要条件, 了解各途径中不同的传输机制, 并将特定的蛋白质基板感兴趣的六个不同的隔间的叶绿体。
分离的膜从叶绿体提供更好的实验控制环境因素 (如盐和基质浓度, ATP/GTP 的存在, 和 pH 条件), 影响测量的运输热力学和动力学。由于同样的原因, 这种体外环境有利于对易位机械细节的探索。此外, 虽然叶绿体蛋白定位的预测软件改善了7,8,体外传输化验提供了一个更快的方法, 以证实显微镜下的荧光化验,需要基因编码的荧光标记, 植物转化和/或特定的抗体。在这里, 我们提出了从豌豆 (豌豆大蒜) 隔离叶绿体和囊体的协议, 以及为每个能量依赖性囊体易位通路优化的运输化验。

<table border="0" cellpadding="0" cellspacing="0" style="width:1936px;" width="1936">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Pisum sativum seeds</td>
			<td style="width:252px;">Seedway LLC, Hall, NY</td>
			<td style="width:292px;">8686 - Little Marvel</td>
			<td style="width:1068px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miracloth</td>
			<td>Calbiochem, Gibbstown, NJ</td>
			<td>475855-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">80% Acetone</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>67-64-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blender with sharpened blades</td>
			<td>Waring Commercial</td>
			<td>BB155S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polytron 10-35</td>
			<td>Fischer Sci</td>
			<td>13-874-617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beckman J2-MC with JA 20 rotor</td>
			<td>Beckman-Coulter</td>
			<td>8043-30-1180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall RC-5B with HB-4 rotor</td>
			<td>Sorvall</td>
			<td>8327-30-1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM dithiothreitol (DTT) in 1xIB</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>12/3/83</td>
			<td>Can be frozen in aliquots for future use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM MgATP in 1xIB</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>74804-12-9</td>
			<td>Can be frozen in aliquots for future use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermolysin in 1xIB (2mg/mL)</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>9073-78-3</td>
			<td>Can be frozen in aliquots for future use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K-Tricine</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>T0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>50-70-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manganese Chloride</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>13446-34-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>115-39-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-Mercaptoethanol</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>60-24-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of physiologically active thylakoids and their use in energy-dependent protein transport assays

叶绿体是绿色植物中的细胞器, 负责执行许多必要的代谢通路, 尤其是光合作用。在叶绿体内, 类囊体膜系统将所有的光合颜料、反应中心配合物和大多数电子载体都安置在一起, 并负责光依赖性 ATP 的合成。90% 以上的叶绿体蛋白被编码在细胞核中, 翻译成细胞质, 随后导入叶绿体。进一步蛋白质运输入或横跨囊状体膜利用四个易位途径之一。在这里, 我们描述了一个高产的方法, 以分离的运输主管类囊体从豌豆 (豌豆大蒜), 连同运输化验通过三能源依赖 cpTat, cpSec1 和 cpSRP 介导的途径。这些方法使实验与囊体蛋白定位, 传输能量学, 和蛋白质易位的机制跨生物膜。

为了测量成功运输的基体的数量, 有一个或多个 "百分比输入" 车道是有用的。对于下面所示的数据, 10% 的最终运输反应没有类囊体使用。这种 "百分比输入" 也有助于可视化前体基底的大小。所述百分比表示已知的、定义的用于比较所述运输基板的基板量, 并可根据需要使用最初制备的蛋白质进行缩放。另外, 建议在 0.75 mm 聚丙烯酰胺凝胶的单一车道上加载少于4µg 的智利当...

Watch this Scientific Journal Video about Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays at JoVE.com

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用

本文提出了用于叶绿体双精氨酸易位 (cpTat)、分泌 (cpSec1) 和信号识别粒子 (cpSRP) 通路的生理活性类囊体和蛋白质转运测定的高产量隔离的协议。

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

这种方法可以帮助回答有关蛋白质在叶绿膜上传输的机制和能量，以及蛋白质在表面细胞中的位置的关键问题。该技术对胸腺蛋白靶向的主要优点是，它允许在运输过程中完全控制胸腺体的环境，包括温度、pH值、离子条件和前体浓度。首先将大约55克豌豆浸泡在400毫升蒸馏水中3小时。将浸泡的豌豆浸泡在土壤中的塑料托盘中，上面覆盖着一层薄薄的紫土，并像文本协议中描述的那样生长9至15天。在叶绿体隔离的当天，在固定角度转子中，将 15 毫升 Percoll 和 15 毫升 2xGB 缓冲液的混合物离心，在 30，900 gs 的固定角度转子中，在 4 摄氏度下，在 4 摄氏度下将 15 毫升的 Percoll 和 15 毫升 2xGB 缓冲液离心，准备连续密度梯度。然后收获约25克9至15天大豌豆芽。将它们添加到 95 毫升的冷 1xGB 缓冲液中，用锐化刀片。和同质化。通过至少两层奶酪布过滤这种混合物，放在冰镇烧杯的漏斗中。在3000克的摆动桶转子中，使用15毫升聚碳酸酯离心管将滤金物离心，在4摄氏度下5分钟。丢弃上一杯，用画笔轻轻将颗粒重新装出1毫升1xGB。之后，使用玻璃姿势移液器小心地将屏幕悬挂分层在 Percoll 渐变的顶部。在4摄氏度下，在8000克的摆动桶转子中离心10分钟，刹车关闭。使用姿势移液器再次小心地将含有完整叶绿体的下部绿带转移到一个新的 50 毫升聚碳酸酯管中，并加入 30 毫升 1xIB 缓冲液。在 1，800 gs 的摆动桶转子中将管在 4 摄氏度下离心 5 分钟。丢弃上一杯后，用画笔轻轻将颗粒重新在1xIB的毫升中。在 1xIB 的 30 毫升中清洗，并在相同条件下再次离心。在1xIB的一毫升最后离心后重新补充颗粒。要确定叶绿素浓度，将10微升完全重新暂停的叶绿体与5毫升80%丙酮混合。通过11微米孔径的180微米厚滤纸进行过滤，然后通过分光光度计确定叶绿素浓度。使用冷1xIB将叶绿体混合物稀释至每毫升1毫克。在不需要频闪提取物时开始分离胸腺素，如cpTat运输，在1.5毫升管中离心完好无损的叶绿体，在4摄氏度的4摄氏度下，在1000克的摆动桶转子中，在6分钟。之后，丢弃上流剂，将颗粒重新在低氧解液缓冲液中，用微管达到每毫升1毫克。在黑暗中冰上孵育，偶尔混合10分钟。为了恢复胸腺，在4摄氏度的摆动桶转子中离心，在4摄氏度下进行8分钟。用一到两毫升的解液缓冲液重新填充颗粒，然后在相同条件下离心两次，清洗胸腺两次。最后离心后，将颗粒重新在1xIB缓冲液的一毫升中重新下水，并使用先前证明的分光光度计确定分离胸腺中的叶绿素浓度。对于需要频闪萃取回恢复的通路，如CPSec1和CPSP在摆动桶转子中分离的600微升叶绿体，在4摄氏度下以1000克离心6分钟。对于叶绿体解，我们用600微升的低质解液缓冲液悬浮颗粒。在黑暗中在冰上孵育10分钟，偶尔混合。然后在3，200克的摆动桶转子中将管子离心，在4摄氏度下8分钟。为了保存频闪，将浅绿色转移到黄色上流液到新的微型离心管中，并添加相等体积的2倍粗频闪缓冲液。要清洗胸腺，在摄氏4度下加入两卷解液缓冲液，并在3，200克下离心，8分钟。将颗粒重新在1xIB中重新浓缩，以达到每毫升1毫克的叶绿素浓度，并放在冰上，供以后用于运输测定。在42，000克的固定角度转子中，在4摄氏度下将稀释的粗频闪离离去30分钟，去除粗频闪中的残余膜。然后从每个管中收集 95% 的体积，确保不打扰黄色小颗粒，同时注意从每个管中取的体积。要准备浓缩的频闪提取物，请将收集的超级天然物集中起来。然后使用四毫升30千吨分子量截止浓缩器浓缩提取物五倍。在冰上1.5毫升管中测定cpTat运输，将分离的胸腺酸混合到每毫升0.33毫克的最终叶绿素浓度、1xIB中准备的5毫摩尔ATP和1xIB中准备的8毫摩尔DTT。要启动运输，请将基底蛋白引入运输组合。在室温下，以每平方米80至100微爱因斯坦/秒的光合作用活性辐射照射悬浮液，进行10分钟的反应。要停止运输反应，用冰 1xIB 稀释悬浮液八倍。在3，200克的微型离心机中，在4摄氏度下5分钟，以回收胸腺。丢弃上清液后，将颗粒重新在120微升的冰冷1xIB中。要测定CPSec1或PSRP通路在1.5毫升的冰上管中混合以下几点。期末叶绿素浓度为每毫升0.33毫克，每毫升叶绿素浓度为0.83毫克，在1xIB中准备5毫摩尔ATP。用冰1xIB将反应达到所需的体积，并在冰上孵育10分钟。启动运输，将基底蛋白的体积为10%的体积引入这种运输混合物。在室温下，以每平方米80至100微爱因斯坦的光合作用活性辐射照射10分钟。为了停止反应，用冰1xIB稀释悬浮液8倍。在3，200g的微型离心机中离心，在4摄氏度下5分钟。丢弃上清液后，将颗粒重新在120微升的冰冷1xIB中。要去除尚未运输的残留基质，在1xIB中加入6微升，每毫升热固辛2毫克，加入10毫摩尔氯化钙，消化外部蛋白质。在冰上孵育这种蛋白酶反应40分钟。在 1xIB 中，用 25 毫摩尔 EDTA 将体积翻倍，使蛋白酶淬火。在3，200克的微型离心机中，在4摄氏度下5分钟，以回收胸腺。取出上一杯后，在1xIB中，在5毫摩尔EDTA的120微升中重新获得胸腺科伊德，并转移到一个新的1.5毫升管。在3，200克的微型离心机中再次离心5分钟后，在4摄氏度下丢弃上流液，将颗粒重新在适当体积的莱姆利样品缓冲液中，辅以10毫摩尔EDTA。将样品放入沸水浴池中 10 分钟。继续按 SDS 页面分析示例。iOE17蛋白质通过cpTat通路运输，导致在引入的基质和成熟加工形式之间发生2至3千吨的大小变化。这是因为n终端信号肽的裂解，表明CPTat传输成功。热解素治疗只留下成功运输，因此蛋白素耐耐成熟带。通过CPSec1通路运输OE33前体，导致成熟基材的大小变化，以及运输基材的蛋白保护。通过cpSRP通路将LHCP前体插入胸腺膜，通过热固辛消化进行评估，导致大小变化为1.5至2千元。这表明膜插入是成功的，因为膜保护成熟的LHCP降解产物的未解，从完整的蛋白解。看完这个视频后，你应该有一个很好的理解，如何隔离胸腺科伊德和通过能量依赖cpTat，cpSec 1和cpSRP通路检测转位。一旦掌握了这项技术，可以在三到四个小时内完成，当执行得当。虽然本视频中没有说明，但隔离和使用光合作用活性叶绿体和胸腺体时，我们通常会在实验室中关闭头顶灯。在尝试这个程序时，重要的是保持隔离的叶绿体和胸腺体在四摄氏度。记得在开始时轻轻重新暂停叶绿体。避免在第一个离心步骤后干扰 Procoll 梯度。此外，百分比输入样品可以与运输反应同时制备，并在同一凝胶上运行以估计基板的传输。不要忘记，使用放射性基材需要认真处理和适当的 PPE。在它的发展之后，这项技术为蛋白质靶向领域的研究人员铺平了道路，以探索能量、动力学和控制蛋白质在胸腺膜上运输的独特机制。

Research

JoVE Journal

Biochemistry

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

科研

生物化学

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...