Name: isolation and characterization of intact phycobilisome in cyanobacteria
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作者感谢国立台湾大学生命科学学院Technology Commons对超速离心机的方便使用。蓝藻菌株 Synechocystis sp. PCC 6803和 Leptolyngbya sp. JSC-1分别由台湾中央研究院的Chu，Hsiu-An博士和美国宾夕法尼亚州立大学的Donald A. Bryant博士赠送。这项工作由科学技术部（台湾）（109-2636-B-002-013-和110-2628-B-002-065-）和教育部（台湾）玉山青年学者计划（109V1102和110V1102）资助。

胞藻属PCC 6803，模型葡萄糖耐受菌株，从台湾中央研究院的Chu，Hsiu-An博士获得。Leptolyngbya sp. JSC-1，非模型丝状，是从美国宾夕法尼亚州立大学的Donald A. Bryant博士那里获得的。
1. 细胞培养和收获
<ol><li>使用金属接种回路将Syn6803或JSC-1细胞接种到含有50 mL B-HEPES培养基的100 mL锥形烧瓶中28。在30°C下，在50μmol光子m-2 s-1（白...

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该协议描述了一种简单而标准的方法，用于分离两种类型的蓝藻中的完整PBS，单细胞模型Syn6803和丝状非模型JSC-1。该方案的关键步骤是在蔗糖的不连续密度梯度上进行细胞均质化和超速离心。通常，丝状细胞的破坏比单细胞细胞更复杂。增加起始物质的量（细胞沉淀的湿重）和重复的磁珠打有助于提高丝状蓝藻细胞PBS的产量。对于丝状蓝藻JSC-1，使用的细胞是单细胞Syn6803的三倍。此外，要完全破?...

藻胆体（PBS）是一种巨大的水溶性色素 - 蛋白质复合物，附着在蓝细菌的类囊体膜中光系统的细胞质侧1。PBS主要由有色藻胆蛋白和无色连接蛋白组成1，2。藻胆蛋白可分为四大类：藻红素、藻红蓝蛋白、藻蓝蛋白和异体花青素3。四大类吸收490-650nm范围内不同波长的光能，叶绿素吸收效率低下3。PBS可以用作光收集天线，用于收集光能并将其输送到光系统II和I4。
PBS的结构和组成因物种而异。总的来说，在不同的蓝藻物种中已经鉴定出三种形状的藻胆体（半盘状、束状和杆状）5。即使在同一物种中，PBS的组成也会随环境而变化，例如光质和营养消耗6，7，8，9，10，11。因此，从蓝藻中分离PBS的实验程序有助于研究PBS12。几十年来，许多不同的方案已经分离出PBS并分析了其结构，组成和功能6，7，8，12，13，14，15，16，17。PBS分离方法种类繁多，确实为使用不同的试剂和仪器分离不同物种中的复合物提供了灵活性。然而，这也使得不熟悉蓝藻和PBS的科学家更难选择合适的方案。因此，在这项工作中为那些有兴趣从蓝藻中开始PBS分离的人开发了一个通用且直接的方案。
此处总结了从以前的出版物中分离 PBS 的方法。由于PBS是一种水溶性蛋白质复合物并且容易解离，因此在提取过程中需要高离子强度的磷酸盐缓冲液来稳定PBS18。过去曾发表过几篇描述从蓝藻中分离PBS的方法的研究文章。大多数方法需要高浓度的磷酸盐缓冲液8，14，15，18，19。然而，机械破坏细胞的程序各不相同，例如玻璃珠辅助提取，超声处理20和法式压榨6，8，14。不同的藻胆蛋白可以通过用硫酸铵20 沉淀并通过HPLC21 或色谱柱22纯化来获得。另一方面，完整的PBS可以通过蔗糖密度梯度超速离心轻松分离6，8，15。
在该协议中，使用一种模型蓝藻和一种非模型蓝藻作为PBS分离的材料。它们是模型单细胞葡萄糖耐受 性Synechocystis sp. PCC 6803（以下简称Syn6803）和非模型丝状 Leptolyngbya sp. JSC-1（以下简称JSC-1），分别7，23，24。该协议首先在高离子强度磷酸盐缓冲液中破坏单细胞和丝状蓝藻。裂解后，通过离心收集上清液，然后用非离子洗涤剂（Triton X-100）处理，以溶解来自类囊体膜的水溶性蛋白质。将总水溶性蛋白施加到不连续的蔗糖密度梯度上以分馏PBS。该方案中不连续的蔗糖梯度由四种蔗糖溶液组成，并将完整的PBS分配在蔗糖层的最低部分25中。PBS的完整性可以通过SDS-PAGE，锌染色和77K荧光光谱分析6，7，8，26。这种方法适用于旨在从蓝藻中分离完整PBS并研究其光谱，结构和组成特性的科学家。
此协议有几个优点。（1）该方法是标准化的，可用于从单细胞和丝状蓝藻中分离完整的PBS。大多数文章描述了应用于任何一种类型的蓝藻4，7，8，12，13，14，16，18的方法。（2）该方法在室温下进行，因为PBS在低温下解离19，27。（3）该方法描述了使用珠状敲击剂来破坏细胞;因此，它比高压法国压力机更便宜，更安全，并且其他方法中超声波仪可能造成的听力损伤8，13，14，20。（4）该方法通过蔗糖梯度超速离心分离完整的PBS。通过这种方式，可以根据蔗糖浓度分离具有不同大小和部分解离PBS的完整PBS。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 mm glass beads</td>
			<td>BioSpec</td>
			<td>11079101</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>13 mL centrifugation tube</td>
			<td>Hitachi</td>
			<td>13PA</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>40 mL centrifugation tube</td>
			<td>Hitachi</td>
			<td>40PA</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>8.1875.2500</td>
			<td>for Coomassie Blue staining</td>
		</tr>
		<tr>
			<td>B-HEPES medium</td>
			<td></td>
			<td></td>
			<td>A modified cyanobacterial medium from BG-11 medium</td>
		</tr>
		<tr>
			<td>Brilliant Blue R-250</td>
			<td>Sigma</td>
			<td>B-0149</td>
			<td>for Coomassie Blue staining</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Wako pure chemical industries</td>
			<td>2-291</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Radwag</td>
			<td>WLC 2/A2/C/2</td>
			<td>for the wet weight measurement of cell pellets</td>
		</tr>
		<tr>
			<td>Fluorescence spectrophotometer</td>
			<td>Hitachi</td>
			<td>F-7000</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>BioShop</td>
			<td>Gly001.500</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>High-Speed refrigerated centrifuge</td>
			<td>Hitachi</td>
			<td>CR22N</td>
			<td>for buffer exchange</td>
		</tr>
		<tr>
			<td>Leptolyngbya sp. JSC-1</td>
			<td></td>
			<td></td>
			<td>from Dr. Donald A. Bryant at Pennsylvania State University, USA.</td>
		</tr>
		<tr>
			<td>Low temperature measurement accessory</td>
			<td>Hitachi</td>
			<td>5J0-0112</td>
			<td>The accessory includes a transparent Dewar container for 77K fluorescence spectra</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>1.07018,2511</td>
			<td>for Coomassie Blue staining</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher</td>
			<td>Pico 21</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Mini-Beadbeater-16</td>
			<td>BioSpec</td>
			<td>Model 607</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>PanReac AppliChem</td>
			<td>121512.121</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>PanReac AppliChem</td>
			<td>141509.121</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Screw cap vial</td>
			<td>BioSpec</td>
			<td>10832</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>SmartView Pro Imager</td>
			<td>Major Science</td>
			<td>UVCI-2300</td>
			<td>for Znic staining signal detection</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Zymeset</td>
			<td>BSD101</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Zymeset</td>
			<td>BSU101</td>
			<td>for PBS isolation</td>
		</tr>
		<tr>
			<td>Synechocystis sp. PCC 6803</td>
			<td></td>
			<td></td>
			<td>glucose-tolerant strain from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BioShop</td>
			<td>TRS 011.1</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioShop</td>
			<td>TRX 506.500</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Ultra 10 K membrane centrifugal filter</td>
			<td>Millipore</td>
			<td>UFC901024</td>
			<td>for buffer exchange</td>
		</tr>
		<tr>
			<td>Ultra 3 K membrane centrifugal filter</td>
			<td>Millipore</td>
			<td>UFC500324</td>
			<td>for buffer exchange</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Hitachi</td>
			<td>CP80WX</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>UV/Vis spectrophotometer</td>
			<td>Agilent</td>
			<td>Cary 60</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>PanReac AppliChem</td>
			<td>131787.121</td>
			<td>for Znic staining</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>BioBasic</td>
			<td>MB0338</td>
			<td>protein loading buffer</td>
		</tr>
	</tbody>
</table>

isolation and characterization of intact phycobilisome in cyanobacteria

在蓝藻中，藻胆体是一种重要的天线蛋白复合物，它收集光并将能量转移到光系统I和II进行光化学。研究藻胆体的结构和组成对科学家来说非常有趣，因为它揭示了蓝藻中光合作用的进化和分化。该协议提供了一种详细且优化的方法，通过磁珠打浆机以低成本有效地破坏蓝藻细胞。然后，完整的藻胆体可以通过蔗糖梯度超速离心从细胞提取物中分离出来。该方法已被证明适用于具有不同细胞类型的模型和非模型蓝藻。还提供了分步程序，通过77K荧光光谱和SDS-PAGE用硫酸锌和考马斯蓝染色来确认藻胆蛋白的完整性和性质。分离出的藻胆体也可以进行进一步的结构和成分分析。总体而言，该协议提供了一个有用的入门指南，使不熟悉蓝藻的研究人员能够快速分离和表征完整的藻类化合物。

将Syn6803和JSC-1细胞在锥形烧瓶中培养，在30°C的B-HEPES培养基中，在LED白光（50μmol光子m-2s-1）下在充满1%（v / v）CO2的生长室中持续搅拌。在指数生长阶段（OD750 = ~0.5），将细胞转培养到最终光密度为OD 750 = ~0.2的新鲜培养基中。在达到晚期指数生长阶段（OD750 = 0.6-0.8）后，收集培养物并离心（室温下10，000×g20分钟）。弃去上清液，并将细胞沉...

Watch this Scientific Journal Video about Isolation and Characterization of Intact Phycobilisome in Cyanobacteria at JoVE.com

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

目前的方案详细介绍了通过不连续蔗糖密度梯度离心从蓝藻中分离藻胆体的过程。完整的藻糖体的分数通过77K荧光发射光谱和SDS-PAGE分析得到证实。所得的藻胆体组分适用于TEM的负染色和质谱分析。

蓝藻中完整藻胆体的分离与表征

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

这是一个简单而快速的方案，允许不熟悉蓝藻的人轻松分离藻类动物。这种技术的优点是它适应了小规模，并且易于执行。它是分离模型和非模型性蓝霉菌完整藻属的简单、可靠和有效的方法。首先将750纳米处0.8 OD的细胞培养物转移到一升烧瓶中，并将其稀释在500毫升B-HEPES培养基中，以达到750纳米处的OD为0.2。在30摄氏度下持续搅拌培养物，直到OD达到0.8比1，表明指数生长阶段较晚。在室温下以10， 000次g的速度离心培养物20分钟并弃去上清液。悬浮细胞沉淀并用0.75摩尔磷酸钾缓冲液洗涤两次，pH值为7。通过在室温下离心将细胞沉淀在50毫升离心管中。弃去上清液并用0.75摩尔磷酸钾缓冲液重悬细胞。通过电子天平测量沉淀的湿重，并将沉淀的一克湿重重在五毫升的缓冲液中。将一毫升细胞悬浮液和0.4至0.6克0.1毫米玻璃珠加入两毫升螺旋盖小瓶中。用珠子打浆机将细胞破碎30秒。让管子在室温水浴中冷却两分钟。裂解后，在室温下以500倍g的速度离心小瓶10秒。用移液管将上清液收集到15毫升的离心管中。用0.5毫升0.75摩尔的磷酸钾缓冲液洗涤珠子一次，然后转移到同一离心管中。将Triton X-100加入裂解的细胞悬浮液中，并在室温和40RPM下在摇摆振荡器上孵育20分钟或直到溶液变均质。将试管以17，210倍g离心20分钟，并将上清液在室温下储存没有上Triton胶束层长达一小时。在pH值为7的0.75摩尔磷酸钾缓冲液中制备四种浓度的蔗糖。将2.8毫升两摩尔蔗糖缓冲液置于40毫升离心管的底部，并用三层蔗糖溶液覆盖。最后，加入三毫升含有上清液馏分的PBS，并离心所得梯度。超离心后，在层之间凝结分馏的PBS和藻胆蛋白，并观察Syn6803和界面中的蓝色条带。通过反复浓缩并用0.75摩尔磷酸钾缓冲液稀释馏分并膜离心过滤单元来除去蔗糖。为了测量PBS荧光，用0.75摩尔磷酸钾缓冲液稀释浓缩的PBS样品，以构成高达500微升的体积。将稀释的PBS样品加入透明玻璃管中，并将管子冷冻在液氮中直至完全冻结。将冷冻管移动到预先装满液氮的透明杜瓦瓶容器中。完全细胞裂解显示离心前的上清液和深蓝绿色。分离的PBS的蔗糖密度梯度分离显示解离藻胆蛋白的几个部分，最低部分代表完整的PBS。JSC one和Syn6803的解离和完整PBS的吸收光谱在622纳米处具有相同的峰值，对应于藻蓝蛋白。此外，在JSC-1的解离和完整PBS中，藻红素吸收峰为571纳米。解离PBS的发射光谱在Syn6803和JSC-1中具有约650纳米的强峰，代表藻蓝蛋白的发射。荧光发射最大峰值在651纳米和684纳米处表明，藻蓝蛋白收集的能量被有效地转移到核心中的别异胞菌蓝蛋白和末端发射器，即ApcD和ApcE。该图显示了紫外照射下的锌染色SDS-PAGE，其中藻胆蛋白亚基具有强烈的荧光，而ApceE显示出微弱的荧光，用箭头表示。Kumasi蓝色染色显示上部部分含有其他非PBS水溶性蛋白，完整的PBS和Syn6803显示出突出的ApcE条带，用箭头表示，缺乏解离的PBS级分。超速离心表明，以1：3的比例制备的蔗糖梯度比以1：10的比例制备的蔗糖梯度显示出更宽的条带。重要的是裂解细胞并完全溶解上清液以获得更多的藻胆体。其他方法，如冷冻电镜或混合光谱法，可用于研究藻糖体的蛋白质结构或蛋白质组成。

Research

JoVE Journal

Biology

Isolation and Characterization of RNA-Containing Exosomes

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

科研

生物学

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Isolation and Characterization of Microvesicles from Peripheral Blood

从外周血中分离和鉴定的微泡

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

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 ...

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...