我们感谢阿迪尔. 艾哈迈德在 FCP 净化方面的帮助。克劳迪娅教授 Büchel 被公认为有帮助的讨论和阅读手稿。这项工作得到德国研究基金会 (DI1956-1/1) 和洪堡基金会 Feodor Lynen 奖学金的支持。

注:FCPs 等光合复合物极易受到光和热的影响。总是在冰和昏暗的灯光下工作。
1. 从细胞中分离 FCP
<ol><li>
从C. meneghiniana细胞分离的类囊体
	<ol>
<li>在五500毫升的烧瓶中生长meneghiniana , 每个填充300毫升的 ASP 中23,25和5000万细胞。用一个棉花塞子塞进烧瓶, 让细胞生长到指数生长阶段大约一个星期在 120 rpm 与 16 h 光和 8 h 黑暗的阶段在40µmol 光子或 (m²s) 白色光和温度在15-18 °c 之间。检查细胞数是在 1.5-200万细胞/毫升之间的细胞计数器室。</li>
		<li>在高速离心机中, 用500毫升离心瓶 (4 °c) 在 4000 x g的预换热器转子上离心出15分钟的电池。用吹打重新悬浮12毫升均匀缓冲中的细胞颗粒 (HB,表 1)。<ol>
<li>将悬浮液转移到一个50毫升的塑料管上。将样品贮存在摄氏-80 摄氏度或进行步骤1.1.3。</li>
		</ol>
</li>
		<li>预冷的珠磨和设备。用玻璃珠混合料将50毫升烧杯填充到 75%, 并添加细胞悬浮液。对于细胞中断, 使用 7 x 四十五年代脉冲在全速与三十年代的冷却之间的每个脉冲。在步骤1.1.6 中采取20µL 破坏细胞进行质量检查。</li>
		<li>在玻璃过滤器漏斗上过滤被破坏的细胞, 然后通过将 HB 浇在玻璃珠上, 直到它们看起来清晰为止。将洗涤分数与滤液一起池。保持最终体积低于150毫升。</li>
		<li>用三50毫升塑料管在 140 x g上离心样品15分钟, 以颗粒状细胞碎片。小心地将上清液转移到20毫升聚碳酸酯离心瓶并丢弃颗粒。<ol>
<li>用 HB 填充小瓶, 平衡重量, 离心机在合适的转子1小时在 30万 x g和4°c, 以颗粒状囊状体膜。</li>
		</ol>
</li>
		<li>使用离心时间检查被破坏的细胞的比例, 显微镜下400X 放大20µL 样本在1.1.3。计算叶绿体游离与叶绿体含 frustules (二氧化硅壳) 的比值。 注意: 硅藻细胞壁是由二氧化硅制成的, 在显微镜中可见为高度衍射的物质。如果细胞中断工作, 叶绿体就应该从细胞中释放出来。</li>
		<li>用小画家的刷子重新悬浮膜小球, 尽可能少的洗涤缓冲器 (0.5-1 毫升)。填充聚碳酸酯离心瓶与洗涤缓冲 (表 1), 平衡他们的重量和离心机为20分钟在 20万 x g和4°c。<ol>
<li>用画家的刷子将洗过的膜重新挂起。添加洗涤缓冲器, 只有当需要保持尽可能高的类囊体浓度。池所有类囊体在一个样品瓶 (15 毫升)。</li>
		</ol>
</li>
		<li>用样品的10µL 和90µL 100% 丙酮预稀释样品。离心机以 1.2万 x g为5分钟, 颗粒沉淀蛋白。采取10µL 的 predilution 和混合它与990µL 90% 丙酮。<ol>
<li>测定叶绿素 a 和 c 的吸光度 (ABS) 在664毫微米和630毫微米在90% 丙酮。从两个值中减去 ABS750nm 。使用以下公式确定总叶绿素含量:24 1)<img alt="Equation 1" src="/files/ftp_upload/58017/58017eq1.jpg"> 2)<img alt="Equation 2" src="/files/ftp_upload/58017/58017eq2.jpg">
</li>
		</ol>
</li>
		<li>整除类囊体0.5 毫克的总叶绿素在1.5 毫升反应管, 冻结他们在液氮和储存在-80 °c, 直到进一步使用。</li>
	</ol>
</li>
	<li>
FCP 配合物的分离与浓缩
	<ol>
<li>准备一个蔗糖梯度溶液和填充 ultracentrifuge 管, 直到顶部减去加载量 (300-500 µL)。将管子冷冻-20 摄氏度, 直到完全凝固。允许管解冻在 +4 摄氏度, 这需要3-4 小时的17毫升管。<ol>
<li>重复冻结解冻循环两次, 以细化渐变以提高分辨率。</li>
		</ol>
</li>
		<li>使用1.1.9 中获得的0.5 毫克叶绿素的样品, 用缓冲 B1 (表 1) 调整成2毫升的最终体积。为增溶, 添加 n-月桂-maltopyranoside (b) 的最终浓度为20毫米。<ol>
<li>翻转管3次, 并把它放在冰上20分钟的温柔晃动, 以避免泡沫。离心机为5分钟, 在 1.2万 x g在一个预冷的桌上部离心机在 +4 °c。</li>
		</ol>
</li>
		<li>在渐变上加载上清。如果使用17毫升小瓶, 则不要载入每梯度的总叶绿素的125µg。离心机为22小时在 10万 x g和 +4 °c。<ol>
<li>使用注射器从渐变中恢复所需的褐色 FCP 分数 (图 2A)。采取5µL 整除和稀释它与995µL B1a。</li>
		</ol>
</li>
		<li>测量紫外-可见光分光光度计中 370-750 nm 之间的吸光度 (ABS) 谱。采用半微光学玻璃小试管。<ol>
<li>打开光谱管理器软件, 转到频谱模式并记录基线, 从370到 750 nm, 然后是样本测量。使用以下设置: 扫描速度, 200 nm/分钟;数据沥青, 0.5 毫微米;反应, 中等。</li>
		</ol>
</li>
		<li>用微测量回收样品的体积。通过将恢复的体积增加两倍于 B1 缓冲器 (表 1) 来清洗 FCP 络合物。集中在一个 30 kDa 截止到 1000 x g和 +4 °c 的膜选矿厂到 ABS672nm至少20。 注:在样品室中, 由于胶束富集, b 的浓度可能上升。这可能会导致进一步的 FCP 复合体的增溶!如果需要进一步洗涤步骤, 以消除残余蔗糖, 避免过度增溶通过添加洗涤剂自由缓冲 B1。</li>
		<li>采取20µL 整除的控制。将液体氮气中的样品冷冻, 并将其贮存在-80 摄氏度。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58017/58017fig2.jpg"> 图 2: 纯化 FCP, 光谱控制和纯度检查.(A)一夜间离心后蔗糖密度梯度的典型出现。所有褐色带都包含由 FCPa 和 FCPb 组成的 FCP 池. pigm.-未绑定颜料, PS--高等植物(B)采用 30 FCP 截止率的离心过滤装置, 在 (蓝线) 和后 (橙色虚线) 浓度下的吸光度谱.特别是, 类胡萝卜素容易从 FCP 的损失, 这将导致较低的吸光度在该地区之间500-550 毫微米。图被规范化对叶绿素 Qy最大值在 ~ 670 毫微米。(c)叶绿素 a (465 nm) 激发的发光光谱, 用于测试函数激发能量转移。如果智利 c 的能量转移受到阻碍, 则会出现一个额外的荧光带, 在 640 nm (智利 c)。图被规范化到发射最大值。(D)在 675 nm (智利荧光最大值) 上记录的激发谱, 用于测试从 370 nm 到 600 nm 之间吸收的所有颜料的能量转移。如果能量转移到智利 a 是低效率的, 荧光率将减少特别是在465和550毫微米之间。图被规范化了到最大大约440毫微米。如果浓度良好, 则 (B)、(C) 和 (D) 中的光谱几乎相同。(E)使用三 tricine 凝胶28检查隔离 FCP 的纯度。FCPa 和 FCPb 在 18-19 kDa 之间有亚基。所有可见的银染色蛋白大于 20 kDa 是污染物。Thyl。-类囊体<a href="https://www.jove.com/files/ftp_upload/58017/58017fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<ol start="3"><li>
基于光谱和凝胶的控制
	<ol>
<li>在1.2.5 后的995µL 中, 记录 370-750 nm 5 µL FCP 的吸收率。将其与步骤1.2.4 中获得的频谱进行比较。使用与步骤1.2.4 中所述相同的检测和设置。<ol>
<li>将数据导出为 csv 并将数据导入电子表格。将两个光谱正常化为约 672 nm 的 Qy波段中的最大 a, 如图 2B所示。</li>
		</ol>
</li>
		<li>通过将所测量的 abs672nm除以所需 abs672nm 0.03, 计算出步骤1.2.4 和1.3.1 中样品的稀释系数。相应地稀释样品与 B1a 并且转移他们到特别玻璃荧光小试管。</li>
		<li>记录 spectrofluorometer 的荧光发射光谱, 以显示从 c 到智利的完整的光能量传递 (图 2C)。<ol>
<li>使用光谱仪软件, 进入频谱测量模式, 具有以下设置: 模式, 发射;狭缝宽度激发和发射, 3 毫微米;灵敏度, 中等;扫描速度, 100 nm/分钟;数据沥青, 0.5 毫微米;激发波长, 465 毫微米;发射, 600-800 nm。执行 autozero 和测量。</li>
		</ol>
</li>
		<li>记录励磁谱与相同的样品和设备, 以揭示完整的能量转移从所有颜料到智利 a (图 2D)。将设置更改为: 模式, 激发;发射波长, 675 毫微米;励磁, 370-600 毫微米。记录光谱。<ol>
<li>用相同范围内的罗丹明光谱校正灯的光谱特性- cf。用户手册中的说明。</li>
		</ol>
</li>
		<li>混合 FCP 样品对应于1µg 的智利 a 与10µL 的 SDS 加载缓冲区。孵育10分钟, 在25摄氏度。离心机为5分钟, 在 12000 x g 的台面上离心机。<ol>
<li>在三 tricine 凝胶上装上上清液28。分开它为 2 h 在 150 V 和银色污点它在分离40以后。 注: FCP 亚基分为两个显著的蛋白带之间的 18-19 kDa, 这是成分的 FCPa 和 FCPb29 (图 2E)。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 脂质体的制备和 FCP 的加入
<ol><li>
脂质混合物和脂质洗涤剂胶束的制备 注意:脂类易受温温度和氧化条件的影响。试着保持脂肪冷冻和2的气氛。<ol>
<li>根据 Vieler等200730, 计算meneghiniana所需的囊体脂质比值。请参阅补充表 1中给出的示例。准备由制造商推荐的脂质库存解决方案在一个溶剂证明容器。</li>
		<li>在2毫升的反应管中吸出所需的脂质, 用温和的氮气流蒸发氯仿, 并尝试将脂质分布到管基的整个区域。让 N2流动, 直到所有溶剂被蒸发。</li>
		<li>溶解29µL 中的脂质混合物, 在4摄氏度的 glucopyranoside 溶液中, 4 小时, 在30摄氏度孵育脂质混合物10分钟。在 3 x 3 分钟的 sonicator 浴中孵化脂质, 在三十年代在冰上中断25摄氏度。<ol>
<li>添加221µL tricine 缓冲液和250µL 的4x 透析缓冲液。</li>
		</ol>
</li>
		<li>使用0.1 µm 聚碳酸酯膜的挤出机, 用于定义的脂质体直径为 50-70 nm。将挤出机与膜和过滤器配套组装。避免气泡, 彻底拧紧总成。</li>
		<li>用4x 透析缓冲液和预湿挤出机填充一支注射器, 直到第二个注射器中没有气泡。</li>
		<li>将脂质洗涤剂胶束应用于挤出机上, 然后将溶液从一把注射器压向另一支。重复此步骤5次, 直到解决方案出现同构。 注:此解决方案可存储在4摄氏度, 数天。不要冻结!</li>
	</ol>
</li>
	<li>
FCP 配合物的加入和洗涤剂和骨料的清除 注:在这个例子中, 我们使用的脂/智利的比率为 12:1, 这相当于一个脂/蛋白质比约100:1。<ol>
<li>添加 FCP 等于20µg 的500µL B1a 缓冲器, 以 250 μL 的挤压脂胶束和250µL 4x 透析缓冲。3 x 3 分钟的样品孵化在25摄氏度的 thermomixer 在 1500-3000 rpm 中断了三十年代暂停冰。</li>
		<li>将四1.5 毫升反应管的盖子切开, 在顶部给出一个仍然适合盖子的环。准备1.5 厘米 x 1.5 厘米的透析膜, 并在20毫升的1x 透析缓冲液中冲洗。</li>
		<li>在每个盖子上填充250µL 的样品。小心, 把膜放在盖子上, 这样车厢就完全充满了样品, 没有气泡发生。拧紧组件上的反应管环, 以便有一个封闭的隔间。</li>
		<li>透析50毫升的1x 透析缓冲液在一个翻滚的振动筛上的冰上过夜 (12-16 小时)。用新鲜的透析缓冲液更换, 再加入7毫克的吸附剂珠, 以除去剩下的洗涤剂至少6小时。</li>
		<li>再次更换透析缓冲器, 再透析12小时. 用200µL 微尖刺穿透析膜, 从反应管盖中吸取所有脂质体, 以恢复脂质体。</li>
		<li>可选步骤: 如果需要高纯度 (> 95%)。在透析缓冲液中, 用含有6%、10%、15% 和20% 蔗糖 epichlorhydrin 共聚物的步骤, 制备17毫升离心瓶中的不连续密度梯度。在摆动斗转子上, 将脂质体放在顶部, ultracentrifuge 在 10万 x g 处, 4 小时。<ol>
<li>用注射器恢复上褐色带, 用 DP 稀释样品 1:5, 然后继续下一步。</li>
		</ol>
</li>
		<li>离心 FCP 脂质体在至少2毫升1x 透析缓冲1.5 小时在 10万 x g 和4°c。在45°角旋转离心管, 以恢复脂质体。允许脂质体向下移动1分钟 (图 3A)。</li>
		<li>恢复 FCP 脂质体的最终体积为25-50 µL. 避免扰乱沉淀。</li>
	</ol>
</li>
	<li>
控制 1: 吸光度, 荧光光谱
	<ol>
<li>添加一个3µL 整除的 FCP 脂质体到1毫升最终容积1x 透析缓冲和离心机5分钟在 1.2万 x g。<ol>
<li>用1.2.4 中描述的相同设备记录 FCP 脂质体370和 750 nm 之间的吸光度。将光谱正常化为 670-680 nm 的 Qy区域的最大值, 并将其与可溶性 FCP 的归一化光谱进行比较 (图 3C)。</li>
		</ol>
</li>
		<li>准备 FCP 脂质体在1毫升的 DP 与吸光度 (ABS) = 0.03 关于最大值介于670-680 毫微米之间。将洗涤剂中的可溶性 FCP 从步骤1.2.6 调整为与 B1a 稀释的同一 ABS。</li>
		<li>记录1.2.4 中所述两种样品的吸光度谱。记录1.3.3 中描述的两个样品的荧光发射光谱。 注:FCP 脂质体样品中荧光率降低 (图 3D和cf )。</li>
	</ol>
</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58017/58017fig3.jpg"> 图 3: FCP proteoliposomes 的分离, 其次是光谱控制和共焦成像。(A)离心后 FCP 脂质体的回收。将离心管转到45°并等待约1分钟-脂质体将向下移动, 而 FCP 骨料未纳入脂质体坚持管壁。(b ) 可溶性 FCP 在脂质体 (橙色) 和 FCP 中的吸光度谱的比较 ( b) 在红色区域 (~ 670 nm-Qy 峰值) 正常化为智利最高值的相同光谱;可溶性 FCP 在洗涤剂 (蓝色) 和 FCP 脂质体 (橙色)。潜在的, 可能是色素的损失, 主要是在 500-550 nm 区域可见的类胡萝卜素。FCP 在脂质体中的聚类可能导致峰值的扩大和智利的轻微移位 (约670毫微米) 到红色。(D)脂质体中洗涤剂和 FCP 中可溶性 FCP 的排放谱。FCP 在脂质体中的聚类增强了 FCP 配合物的能量相互作用, 降低了荧光的产生率 (橙色曲线), 并将发射极大值略微移到红色。<a href="https://www.jove.com/files/ftp_upload/58017/58017fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

作者没有什么可透露的。

FCP 脂质体具有天然脂质成分, 为研究其体外光谱特性提供了方便、简便、重现性的工具。FCP 脂质体的脂质环境类似于类囊膜内的情况, 导致实验结果更接近自然条件。
利用meneghiniana作为 FCP 天线的模型系统具有多种优点。它的生长相对较快, 比其他硅藻模型物种更健壮,例如海链藻 pseudonana。在珠磨中, 细胞断裂相对容易, 这使得完整的囊状体复合物的高产。...

像硅藻这样的光合有机体必须应对不断变化的光照条件, 并应对复杂的适应机制, 保持高光合效率和保护光氧化损伤所造成的过度光照。光合真核生物的一个主要的光保护过程是高能淬火 (qE) 的吸收光作为主要贡献的非光化学淬火 (NPQ) 在轻应力条件下1,2 ,3。光采集天线配合物 (LHC) 参与了励磁能量传递通路的调节。由于在叶绿体腔内高光诱导低pH 值, 天线系统从光收获状态切换到淬火状态。这种能量耗散状态保护-高等植物 (PS) 和其他配合物在类囊体膜从光氧化。在光合真核生物中, qE通常由两个因素1、2、3引起。一个因素是专门的光收获蛋白对低pH 值的反应。公安局蛋白在高等植物中诱导 qE 4。LhcSRs5, 由公安局活动调制, 诱导绿藻中 qE 6。硅藻具有与 LHCSRs7、8、9、10的结构相关的 Lhcx 蛋白。
qE的第二个因素是叶黄素的周期, 其中的类胡萝卜素被转化成光保护形式, 通过环氧化和还原环氧化。在植物和绿藻中, 紫黄素转化为玉米黄质。在硅藻中, diadinoxanthin 转换为 diatoxanthin, 然后与 NPQ11的程度相关。硅藻光收获天线具有一定的特殊性, 尽管它与植物和藻类 LHCs 有关。从光照到光保护的转换速度非常快, 与植物12相比, NPQ 容量更高。这可能是为什么硅藻在不同的生态环境中非常成功的原因之一, 他们负责45% 的大洋净初级产量13。因此, 硅藻光收获系统是光合作用研究的一个有趣的对象。
硅藻, 像中心物种Cyclotella meneghiniana,拥有类囊体固有的光采集系统, 命名后的颜料, 他们绑定-fucoxanthin, 叶绿素 (智利) a 和 c, 因此 FCP. 光收获蛋白, 如 FCPs, 是嵌入在囊体膜系统中, 包括几个膜层。硅藻形成三类囊体的带状。这种复杂的情况使得研究它们在类囊体膜中的分子水平是困难的。此外, 许多组件有助于调节光的收获 (见上文)。因此, 在许多方法中, 复合体是从膜中分离出来的, 使用温和的洗涤剂, 如 n-月桂-maltopyranoside (β), 溶解膜, 但保持 FCP 配合完好。利用可溶性 FCP 对分子内能量转移14、15、16、17进行了多光谱研究。然而, 这种前一种方法是有限的, 因为调节能量转移需要激子吸收与其他天线配合物或-高等植物的相互作用。因此, 这些类型的研究不能与可溶性配合, 因为复合体之间的相互作用失去了。
天线调节的一个重要特征是类囊体膜18中天线和-高等植物的 "分子拥挤"。以前, 一种简单的方法来模拟这种效应的体外.洗涤剂被去除, 导致天线复合体的随机聚集。虽然这一方法17、19获得了一些合理的数据, 但洗涤剂的去除并没有反映体内的情况, 也有一些局限性, 因为复合物在其正规的第三系中没有相互作用。结构。
脂质体的使用克服了前几个局限性。第三级结构仍然完全完好。脂质体膜为天线配合物提供了准本机环境。膜从外部环境中分离脂质体的内部。通过这些方法, 脂质体提供了两个反应室, 用于研究离子和 pH 梯度以及运输过程。此外, 实验系统的参数可以更容易地控制在类囊体膜的研究。脂质体已经被证明是研究光合络合物的极好工具。过去的一个主要重点是植物 lhc, 在那里, 改变脂质成分的影响测试了 LHC II20。在其他方法中, 研究了不同的 LHC II. 的蛋白质-蛋白质相互作用21。此外, 还对绿藻进行了一些研究, 描述了 LHC22之间的自发聚类。考虑到硅藻在水生生态系统中的重要性, 对硅藻天线配合物进行了比较少的研究。两项研究调查了中心Cyclotella meneghiniana的天线配合物, 其中显示了 FCP 天线23的聚类和 FCP 对电化学梯度24的响应。因此, 脂质体是研究硅藻天线及其在近地环境中相互作用和调节的极好工具。脂质体是多才多艺的, 因为许多条件, 如脂组成, 脂质体的大小, 蛋白质密度和周围的水相可以控制。此外, 该方法需要少量的样本。实验系统鲁棒性强, 重现性高。脂质体的划分允许研究 pH 值和离子梯度, 这是调节天线配合物的重要因素。
在这里, 我们描述了 FCP 天线配合物的分离从C. meneghiniana和他们的纳入脂质体与自然类囊脂组成。同时, 我们为可溶性 FCP 的光谱表征提供了典范的数据, 并与脂质体中的 FCP 进行了比较。该方法总结了从改进 Gundermann 和 Büchel 201223、娜塔莉等201622、艾哈迈德和 Dietzel 201724获得的知识和标准化协议。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58017/58017fig1.jpg"> 图 1: 工作流的示意图表示形式。(1)是指1段, 描述细胞生长, 破坏和类囊体的分离与以下 FCP 分离的蔗糖密度梯度;沙菲·萨米-Cyclotella meneghiniana细胞。(2) 2 段中描述的天然类囊体脂质混合物 (MGDG、DGDG 和 SQDG) 的制备, 并建立 octylglycoside 脂质洗涤剂胶束。所定义的脂胶束大小是通过使用已定义孔径的膜挤压而实现的。FCP 和脂胶束在预定的脂质上统一: 蛋白质比,通过控制透析形成 FCP proteoliposomes 去除清洁剂和β。<a href="https://www.jove.com/files/ftp_upload/58017/58017fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>500 ml centrifuge vials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>high speed centrifuge</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Mill VI 2</td>
			<td>Edmund-B&#252;hler (edmund-buehler.de)</td>
			<td></td>
			<td>newer version: Vibrogen-Zellm&#252;hle Vl 6</td>
		</tr>
		<tr>
			<td>Silibeads S 400 &#181;m</td>
			<td>Sigmund-Lindner.com</td>
			<td>5223-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Silibeads S 1,-1,3 mm</td>
			<td>Sigmund-Lindner.com</td>
			<td>4504</td>
			<td></td>
		</tr>
		<tr>
			<td>VitraPOR filter funnel - por1</td>
			<td>ROBU GmbH</td>
			<td>21121</td>
			<td></td>
		</tr>
		<tr>
			<td>polycarbonate ultracentrifuagtion vials (30 mL) for T-865</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>314348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge Discovery 90SE</td>
			<td>Sorvall</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor T 865</td>
			<td>ThermoFisher Scientific (thermofisher.com)</td>
			<td>51411</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer Cell Counter Chamber (improved)</td>
			<td>Carl Roth Laborbedarf (Carlroth.com)</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Mikroskop Primostar (7)</td>
			<td>Optik-Pro (optik-pro.de)</td>
			<td>51428</td>
			<td></td>
		</tr>
		<tr>
			<td>optical glass cuvettes (6040-OG)</td>
			<td>Hellma Analytics (hellma-analytics.com)</td>
			<td>&#34;6040-10-10&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>V-630 UV-VIS Spectrophotometer (incl. software)</td>
			<td>Jasco (jasco.de)</td>
			<td>V-630</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Dodecyl-&#946;-D-Maltopyranoside</td>
			<td>ANATRACE (anatrace.com)</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear tubes 17 ml for AH629</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>344061</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor AH629-17-mL</td>
			<td>ThermoFisher Scientific (thermofisher.com)</td>
			<td>54285</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane concentrator_Centriprep 30 kDa cutoff</td>
			<td>Millipore (merckmillipore.com)</td>
			<td>4307</td>
			<td></td>
		</tr>
		<tr>
			<td>Biometra Minigel-Twin</td>
			<td>Analytik Jena AG (analytik-jena.de)</td>
			<td>846-010-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain Plus Kit</td>
			<td>Bio-Rad (bio-rad.com)</td>
			<td>1610449</td>
			<td></td>
		</tr>
		<tr>
			<td>libre office spread sheet</td>
			<td>The document foundation</td>
			<td>https://de.libreoffice.org/download/libreoffice-still/</td>
			<td></td>
		</tr>
		<tr>
			<td>special glass cuvettes for fluorescence (101-0S)</td>
			<td>Hellma Analytics (hellma-analytics.com)</td>
			<td>101-10-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrofluorometer FP-6500 (incl. Software)</td>
			<td>Jasco (jasco.de)</td>
			<td>FP-6500</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-loading buffer Roti-Load</td>
			<td>ROTH (carlroth.com)</td>
			<td>K929.1</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octyl &#946;-D-glucopyranoside</td>
			<td>ANATRACE (anatrace.com)</td>
			<td>O311</td>
			<td></td>
		</tr>
		<tr>
			<td>Monogalactosyl Diaclyglycerol (MGDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1300</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>Digalactosyl Diacylglycerol (DGDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1310</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>Sulphoquinovosyl Diacylglycerol (SQDG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>59-1230</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>L-alpha-Phosphatidylglycerol (PG)</td>
			<td>Larodan AB (larodan.com)</td>
			<td>37-0150</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>L-&#945;-Phosphatidylcholine</td>
			<td>Sigma-Aldrich (sigmaaldrich.com)</td>
			<td>P3782 SIGMA</td>
			<td>make stock solution in chloroform</td>
		</tr>
		<tr>
			<td>sonicator bath S-50TH</td>
			<td>Sonicor (getmedonline.com</td>
			<td>SONICOR-S-50TH</td>
			<td></td>
		</tr>
		<tr>
			<td>mini-Extruder</td>
			<td>Avanti Polar Lipids (Avanti.com)</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuleopore polycarbonate membrane</td>
			<td>Avanti Polar Lipids (Avanti.com)</td>
			<td>610005</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis membrane Visking 14 kDa cutoff</td>
			<td>ROTH (carlroth.com)</td>
			<td>0653.1</td>
			<td>boil in destilled water before use</td>
		</tr>
		<tr>
			<td>Biobeads SM2 Adsorbent</td>
			<td>Biorad (Bio-rad.com)</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose epichlorhydrin copolymer - Ficoll 400</td>
			<td>Sigma-Aldrich (sigmaaldrich.com)</td>
			<td>F4375</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuagtion vials (2.7 mL) for TFT 80.4</td>
			<td>Beranek Laborger&#228;te (Laborgeraete-beranek.de)</td>
			<td>252150</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor TFT 80.4</td>
			<td>Millipore (merckmillipore.com)</td>
			<td>54356</td>
			<td></td>
		</tr>
		<tr>
			<td>material listed in order of appearance </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For specific safety instructions please refer to material safety sheets and repective manuals.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard lab material and substances are not listed.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating and incorporating light-harvesting antennas from diatom cyclotella meneghiniana in liposomes with thylakoid lipids

植物、藻类和硅藻的光合性能强烈依赖于叶绿体类囊体膜中的光收获和能量传递过程的快速有效调控。硅藻的光收获天线, 所谓的 fucoxanthin 叶绿素 a/c 结合蛋白 (FCP), 是需要的光吸收和有效地转移到光合反应中心, 以及为光保护从过量的光。这两个函数之间的切换是一个长期的研究课题。其中许多研究都是用 FCP 在洗涤剂胶束中进行的。在相互作用研究中, 洗涤剂被去除, 导致 FCP 复合物的不特异聚集。在这种方法中, 很难区分工件和生理相关数据。因此, 通过研究蛋白质-蛋白质相互作用, 能量转移和其他光谱特征, 如果它们嵌入在他们的本地脂质环境中, 就可以获得更有价值的 FCP 和其他膜结合光采集配合物的信息。其主要优点是脂质体具有定义的大小和定义的脂/蛋白比, FCP 聚类的程度受到控制。此外, 在体内调节光收获的 pH 值和离子成分的变化也很容易被模拟。与类囊膜相比, 脂质体更均匀、不复杂, 便于获得和理解光谱数据。该协议描述了 FCP 分离纯化、脂质体制备、FCP 脂质体与天然油脂成分结合的过程。给出了典型应用的结果, 并进行了讨论。

该协议描述了从Cyclotella meneghiniana的总 FCP 分数的分离, 并将其纳入脂质体与本机脂组成。类囊体的分离是高度重现性的, 但囊体产量可能会改变。如果在步骤1.1.4 中回收超过50% 的颜料, 结果是可以接受的。超过80% 是最佳的。
类囊体的增溶是一个关键的步骤。如果步骤1.2.2 中的上清液含有大部分颜料, 则获得可溶性膜。如...

Watch this Scientific Journal Video about Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids at JoVE.com

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

在这里, 我们提出了一种从硅藻中分离 fucoxanthin 叶绿素 a/c 结合蛋白 (FCP) 的协议, 并将其纳入脂质体和天然脂质成分, 研究了离子成分变化时的激发能量传递。

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

isolating-incorporating-light-harvesting-antennas-from-diatom

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Cell Biology

Biochemistry

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Unit 1

Chloroplasts and Photosynthesis

SCIENTISTS IN ACTION

7.7K 次观看.  Goethe University. 在这里, 我们提出了一种从硅藻中分离 fucoxanthin 叶绿素 a/c 结合蛋白 (FCP) 的协议, 并将其纳入脂质体和天然脂质成分, 研究了离子成分变化时的激发能量传递。

视频: Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

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 ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

完全加工重组KRAS4b:分离和描述脱脂和甲基化蛋白

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The &#8220;tubulin code&#8221; is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs.
To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps.
We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.

通过聚合-去聚合周期，通过有限来源的受控后翻译修改和异种型对图布林进行净化

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...