这一步的方法是由杜普利集团 (英国剑桥大学) 的各个成员开发和优化的, 我们赞赏他们的贡献。这项工作是作为能源部联合生物能源研究所 (http://www.jbei.org) 的一部分, 由美国能源部、科学办公室、生物学和环境研究办公室 (DE-AC02-05CH11231) 在劳伦斯之间签订合同的情况下资助的。伯克利国家实验室和美国能源部。我们还感谢西娅挑选和 Vy 非政府组织帮助准备csla9示例。

1. 植物材料的收割 <ol> <li> 收获新鲜植物材料 (约20毫克), 并立即淹没在 96% (v/五) 乙醇和孵化在70和 #176; C 为 30 min; 这将停用任何细胞壁降解酶的存在。对于干物料, 从步骤2开始. 
	警告: 乙醇是易燃的. 
	注: 单茎或莲座叶将提供足够的材料进行分析。但是, 如果大量的组织被汇集并进行分析, 则会出现更少的错误, 因为这更容易称量和处理. </li>
	<li> 仔细记录组织类型和发育阶段, 因为多糖结构随两者的不同而变化。例如, 用 拟南芥 (此处使用), 根据百 et al. 中的方法对组织进行分期。 15 注意: 在本协议中, 我们使用了花序茎的下半部分, 其中第一丝是完全拉长, 但不泛黄. </li> </ol>
 2. 乙醇不溶残渣 (空气) 的制备 <ol> <li> 准备空气, 按照莫蒂默的方法 et al. 9 或其他类似方法, 以产生缺乏可溶性糖的粉末, 如蔗糖和淀粉. </li>
</ol> 3。空气的 Aliquoting 和前处理 注意: 每个样品所需的空气的确切数量取决于 (a) 物质中的多糖的丰度和 (b) 释放的寡糖的平均大小生长激素。该方法在每个寡糖的还原端添加一个单一的荧光分子, 因此寡糖的数量决定了实验的灵敏度。作为一个指南, 在拟南芥茎消化与 GH5/GH26 (如所述), 500 和 #181; g 是推荐的 11 , 而对于在拟南芥茎中的木聚糖与 GH11, 100 和 #181; g 是建议在莫蒂默和 #39; s 研究 3 . <ol> <li> 将空气 (10-15 毫克) 称重为15毫升离心管, 并将 H 2 O 添加到最终浓度2毫克/毫升。涡达到一个均匀悬浮。如果这是问题, 然后使用玻璃匀浆机来协助这个过程. </li>
	<li> 分500和 #181; g 成离心管。干燥等分使用真空离心机 (参见 材料表 ) 过夜在30和 #176; C. </li>
	<li> 在室温下用1米氢氧化钠 (20 和 #181; L) 处理1小时的空气; 这 de-acetylates 了多糖和膨胀纤维素微, 扰乱了生物量结构, 并允许 GH 进入. 
	注意: 这一步可以跳过纯化多糖。小心和 #8211; 强酸/碱。
	<ol> <li> 包括 no-enzyme 空气控制的分 (与所有示例相同, 除步骤4.1 之外). </li>
	</ol> </li> <li> 添加200和 #181; H 2 O 和20和 #181; l 1 M HCl 以中和。通过去除1和 #181 的方法测试 ph 值为 ~ 6-7; 我把它放在纸上 ph 指示条上. </li>
	<li> 添加50和 #181; l 1 米醋酸铵缓冲液, ph 值 6.0 (或任何 ph 值适用于研究中使用的全球统一制度) 和 H 2 O 以给出总容量500和 #181; l. 
	注: 醋酸铵是使用, 因为它升华后, 干燥, 所以它不会增加额外的盐的样品。过量的盐会导致较差的带状和分辨率的速度凝胶. </li>
</ol> 4。空气水解与糖水解 (全球统一制度) 注意: 正如讨论中所提到的, 全球统一制度的纯度至关重要。仅用 heterologously 表达, 亲和纯化酶。在收到制造商的每一个批次后, 水解等分的定义的基板 (例如, 500 和 #181; g 空气) 随着 GH 过夜量的增加 (如 , 0.5, 1, 2, 5, 10, 15, 20 和 #181; l) (3 单位/和 #181; l)。当有超过 GH, 步伐指纹将看起来相同。由于必须确定水解反应正在接近终点, 因此需要过量的酶来提供可重现的结果. 
<ol> <li> 将 mannanases (GH5 和 GH26 11 ) 的预定数量 (见上图) 添加到从步骤3.5 的缓冲区中的空气等分, 以及一个正控制 (30 和 #181; g) 和一个无空气负控制 (酶混合在空管)。涡旋, 然后短暂旋转, 以收集在管底部的反应混合物. </li>
	<li> 在37和 #176 的夜间孵育; C (或选择适当的温度) 与温和的鼓动 (〜 100 rpm). </li>
	<li> 停止在95和 #176 的孵化反应; C 为 20 min. 
	注: 瓶盖封口可用于封头离心管。
	<ol> <li> 离心机使用台式顶离心, 最大速度为10分钟, 并保留上清液. </li>
	</ol> </li> <li> 250 和 #181 中的重颗粒; L H 2 O, 离心机如上所述, 并保留上清液. </li>
	<li> 将两个清和干燥的真空离心机 (见 材料表 ) 在30和 #176; C (〜3小时或隔夜没有暖气). </li>
</ol> 5。低聚糖标准的制备 <ol> <li> 在 H 中准备 1 mM 库存解决方案 2 甘露糖 (人), mannobiose (人 2 ), mannotriose (人 3 ), mannotetraose (人 4 ), mannopentaose (人 5 ) 和mannohexaose (人 6 ), 全部和 #946;, 1-4 链接。分和存储在-20 和 #176; C 直到需要. </li>
	<li> 准备3不同浓度的一个人 1-6 混合物通过组合1和 #181; l (标准 S1), 2 和 #181; l (S2) 或5和 #181; l (S3) 的所有六. </li>
	<li> 干燥在真空离心机 (参见 材料表 ) 在30和 #176; C (~ 1 h). </li>
</ol> 6。Derivitization 低聚糖 <ol> <li> 在 H 中准备 0.2 M 蚂蚁 (8-萘-13, 6-三酸) 的库存解决方案 2 O: 乙酸17:3。温暖的股票到60和 #176; C 完全溶解固体。存储在-20 和 #176; C, 保护免受光, 为 2-3 月. </li>
	<li> 在亚砜中制备 2-甲基吡啶硼烷 (2-PB) 的0.2 米库存溶液。这是非常吸湿, 所以立即重所有粉末后收到的亚甲基亚砜。分, 并存储在-20 和 #176; C 为 1-2 年。根据需要解冻等分 (存储在4和 #176; C 为2周, 然后丢弃). </li>
	<li> 准备 H 的衍生化缓冲区 2 O: 乙酸: 亚甲基亚砜在 17:3:20. </li>
	<li> 对每个样本, 添加5和 #181; 蚂蚁, 5 和 #181; l 2 PB 和10和 #181; l 衍生化缓冲。简要地在管子的底部旋转收集, 漩涡彻底, 然后简要地再旋转。有关反应描述, 请参见 图 1 . </li>
	<li> 在37和 #176 夜间孵育样品; C, 免受光照. </li>
	<li> 干燥在真空离心机 (参见 材料表 ) 在30和 #176; C (~ 2 h). </li>
	<li> 重100和 #181 中的样品和标准; L 3 米尿素。存储在-20 和 #176; C, 在需要时受光照保护. </li>
</ol> 7。速度凝胶的制备 注意: 凝胶铸造设备的装配将取决于品牌。在这里, 我们 use a 垂直电泳系统 (参见材料目录 ), 装备 18 cm x 24 cm 玻璃板材和1.5 毫米间隔物. 
<ol> <li> 根据制造商和 #39 的说明装配凝胶铸造设备. </li>
	<li> 使10x 速度缓冲器 (1M 三硼酸盐, pH 值 8.2) 如下: 添加121.14 克的三基到〜400毫升 H 2 O, 和混合溶解。通过添加固体硼酸 (约60克) 调整 pH 值至 8.2, 然后使体积达1升. </li>
	<li> 在 H 中进行 10% (含/五) 过硫酸铵 (APS) 库存 2 o. 分和存储在-20 和 #176; c. 解冻等分一次, 存储在4和 #176; c 并在2周后丢弃. </li>
	<li> 制作并倒入溶解的凝胶。对于上述设备, 1 凝胶等同于50毫升。在50毫升离心管, 混合 H 2 O (20.2 毫升), 5 毫升10x 速度缓冲, 24.6 毫升40% 丙烯酰胺/双-丙烯酰胺 (29:1 丙烯酰胺: bis)。(警告: 有毒). 
	<ol> <li> 添加200和 #181; ap 和 20 #181 的 l; n、n、n、n 和 #180;-四甲基乙二胺 (TEMED)。轻轻地倒置混合 (避免引入气泡)。用血清学或其他大容积的吸管将凝胶倒入玻璃盘顶部4厘米以下。注意气泡被困在凝胶中的可能性。如果他们这样做, 停止浇筑, 并倾斜/轻拍凝胶释放它们. </li>
	</ol> </li> <li> 用异丙醇覆盖凝胶 (警告 : 可燃) 或者, 小心地用 H 2 o 允许凝胶聚合 (20-30 分钟), 然后将顶层倒掉。如果使用了异丙醇, 然后用 H 2 冲洗 2x, 用吸水纸擦干任何多余的液体. </li>
	<li> 制作并倒入堆积凝胶。混合6.8 毫升 H 2 O, 1 毫升10x 速度缓冲, 2.8 毫升丙烯酰胺/双-丙烯酰胺 ( 警告: 有毒), 80 和 #181; l APS, 8 和 #181; l TEMED 在15毫升离心管。倒置混合, 并覆盖在聚合解决凝胶顶部。轻轻地插入梳子, 避免在梳齿下捕捉气泡. </li>
	<li> 允许凝胶聚合 (20-30 分钟), 用湿纸巾包裹, 然后在塑料包装, 并存储在4和 #176; C 直到需要。商店与梳子到位. 
	注: 如果保持湿润, 则胶凝剂可贮存最多2周 (虽然少于1周). </li>
</ol> 8。运行速度胶凝体 <ol> <li> 使用永久性标记对玻璃上的井位进行标记, 这将有助于跟踪加载顺序, 并识别出井的位置, 一旦梳子被移除. </li>
	<li> 卸下梳子。用1x 速度缓冲器填充井. </li>
	<li> 使用10和 #181; 微量要加载2和 #181; 标准和样本到井中。避免使用最外面的车道, 这往往是较差的运行样本. </li>
	<li> 组装凝胶运行装置的上部腔室, 并将凝胶放在冷却的 (〜10和 #176; c) 运行槽 (10 和 #176; c) 中, 其中含有1x 速度缓冲器。用1x 的速度缓冲填充上腔. </li>
	<li> 打开电源, 在 200 v (恒定 v) 上运行凝胶30分钟, 然后将电压增加到1000伏, 1 小时40分钟。保护凝胶免受光照 ( 例如, 通过在黑色垃圾袋中包装容器). 
	注: 检查凝胶〜5分钟后, 打开电压, 然后再5分钟后增加电压到 1000 v如果电源包无法保持电压, 则可能出现缓冲区泄漏。从水箱中取出凝胶。重新组装上腔, 仔细检查所有垫圈的位置。一个大的芯片上边缘的玻璃板可以防止板形成一个良好的密封与垫片。这通常可以暂时解决, 添加一滴 5% (瓦特/v) 熔融琼脂糖到玻璃的缺口部分, 然后加入上部腔. </li>
</ol> 9。可视化速度凝胶 注意: 使用 transilluminator 中装有长波紫外线灯泡的凝胶成像系统, 以及适用于荧光的 530 nm 的照相机的发射过滤器。或者, 也可以使用激光扫描仪, 如 Goubet 2 中所述. 
<ol> <li> 确保凝胶成像系统在无灰尘的同时用湿润的无棉组织擦拭. </li>
	<li> 从速度槽中取出凝胶, 并简要查看凝胶仍然在玻璃板 (和 #60; 80 毫秒) 以确定染料前面是否仍在凝胶上. </li>
	<li> 使用楔形工具打开凝胶, 同时凝胶仍然在一个玻璃板上, 使用比萨刀将两个堆叠的凝胶除去, 如果染料前面仍在凝胶上, 则凝胶的底部. </li>
	<li> 把 ~ 5 毫升 H 2 O 到 transilluminator 的表面, 然后将凝胶直接转移到 transilluminator。将滤镜设置为 uv 605, 然后使用该软件打开长波 uv transilluminator (请参见 材料表 ). 
	注: 这里使用的蚂蚁荧光有 353/520 nm 的激发/发射. </li>
	<li> 在不同曝光时间拍摄多个图像 (, 如 100 毫秒到十年代)。通过单击 uv 灯按钮 (关闭) 以避免降低荧光, 确保在图像之间关闭 uv 光。确保至少2的图像没有饱和带 (凝胶成像软件将表明这一点). </li>
	<li> 将文件另存为高分辨率. tif 图像. 
	注: 图像可以使用带有凝胶成像系统的软件进行分析, 或者使用免费的图像分析软件, 如 ImageJ (https://imagej.nih.gov/ij/)。有关定量的讨论, 请参见 结果 一节. </li>
</ol>

<ol>
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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=Growth+stage-based+phenotypic+analysis+of+Arabidopsis:+a+model+for+high+throughput+functional+genomics+in+plants.">Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants.</a> Plant Cell. 13 (7), 1499-1510 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Carbohydrate Active Enzyme Database. , (2017).</li><li>Hogg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+modular+architecture+of+Cellvibrio+japonicus+mannanases+in+glycoside+hydrolase+families+5+and+26+points+to+differences+in+their+role+in+mannan+degradation.">The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.</a> Biochem J. 371, 1027-1043 (2003).</li><li>Brennan, Y., 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=Unusual+microbial+xylanases+from+insect+guts.">Unusual microbial xylanases from insect guts.</a> Appl Environ Microbiol. 70 (6), 3609-3617 (2004).</li><li>Li, 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=Development+and+application+of+a+high+throughput+carbohydrate+profiling+technique+for+analyzing+plant+cell+wall+polysaccharides+and+carbohydrate+active+enzymes.">Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes.</a> Biotechnol Biofuels. 6, (2013).</li></ol>

作者没有什么可透露的。

速度是表征多糖结构的直接方法。它可以适用于任何已知的具有特征的活动的全球统一制度的多糖, 在文献1,6,9,11中看到许多例子。通过利用定义的多糖基质和受体, Tt 也被应用于新的全球统一制度12、13、18和转移9的特性。
可重现、解释的结果取决于三关键步骤。首先, 使用的全球统一制度应该不受污染活动的干扰。这是最好的实现, 只有使用 heterologously 表达, 亲和纯化酶。其次, 对于大多数的实验, 重要的是, 酶水解的基质是在完成。这将确保同一个生长激素水解相同的生物量在每一个实验中给出相同的指纹。最后, 在衍生化反应中应该有过量的荧光。这样可以确保可用的减少端被标记为接近100%。这将导致结果的高重现性, 以及数据的数量。
凝胶和试剂质量是确保可重复数据的关键。质量较差的缓冲器, 特别是不正确的 pH 值, 凝胶中的气泡, 以及过量的盐的样品都能影响寡糖的分辨率和保留因子。但是, 将协议和结果部分中介绍的建议的控件包括在内将启用疑难解答。
其识别低聚糖的能力限制了速度。对于一些实验, 一个简单的指纹是所有必要的。然而, 要真正定量在空气中的葡甘聚糖的数量, 例如, 所有释放寡糖的身份是必需的, 这是一个耗时的过程。对于不太复杂的多糖, 如木聚糖的3, 这种方法更为简单。由于有少量的低聚糖标准可供商业使用, 它可能并不总是可能或可取的, 以确定所有的波段。在这种情况下, 速度可以提供非常免费的数据质谱 (即, MALDI CID9或 ESI-MS7, 和核磁共振6)。对于这些方法, 它通常是有益的做一个 large-scale 的准备, 分离的大小-排除色谱, 然后分析每个分数的两个速度之前的 MS 或核磁共振。虽然据报道, 带可以切除和识别的 MALDI-CID, 在实践中, 我们发现, 这是一个低成功率 (可能由于与丙烯酰胺凝胶的相互作用, 当暴露在紫外线光)。
速度的另一个主要限制是吞吐量。要有良好的质量, 解释凝胶与适当的控制, 每一个凝胶将只有〜10实验样品, 和研究员可以预计运行〜4步伐凝胶每天。最近, 一个版本的速度使用毛细管电泳 (CE) 已开发19, 这使得准备样品在 96-井板。它已成功地用于表征糖酶活性和多糖结构6,19, 虽然它需要访问 CE 机器, 这可能是昂贵的购买和维护。

用凝胶电泳 (速度) 分析多糖的方法是对多糖的详细描述1,2,3。植物细胞壁多糖是众所周知的难以分析, 大多数方法需要昂贵的设备, 熟练的操作员, 和大量的纯化材料。在这里, 我们描述了一种获取详细的多糖结构信息的简单方法, 包括结构异构体的解析。
研究植物细胞壁生物合成或植物细胞壁在植物发育中的作用, 是了解植物细胞壁多糖结构的必要条件。然而, 最近, 植物细胞壁的组成已成为有趣的一个更广泛的研究小组, 由于重点是利用植物生物量 (本质上是细胞壁) 作为原料生产生物燃料和生化4。例如, 有效的酶糖化这一材料需要详细了解的多糖结构, 使优化酶鸡尾酒可以选择5。
速度比其他方法有几个优点, 使其成为复杂糖结构快速分析的理想选择。首先, 它不需要纯化的多糖的问题, 是需要的解决方案状态核磁共振 (NMR)6。其次, 速度可以解决结构异构体, 不像质谱 (MS,例如,基质辅助激光解吸/电离 (MALDI) 和电喷雾电离 (ESI)), 这也可能是挑战, 以执行液相色谱 (LC)7. 第三, 速度是非常敏感的, 与 low-picomole 分辨率, 不像薄层色谱 (TLC) 或纸层析。最后, 它不需要昂贵的设备或专业知识, 如 MS, 核磁共振和 LC 的情况。
这种速度方法依赖于糖水解酶 (GH) 的特异性, 它以多糖的混合物中的某些糖苷联系为目标。当 GH 酶在多糖链上作用时, 它会发现一个还原的末端, 然后可以被化学衍生, 在这种情况下用荧光标签。因此, 此方法无法检测到样本的 unhydrolyzed 部分。用电泳法在大格式丙烯酰胺凝胶中分离出标记的寡糖。这给非常相似的分子的卓越的决议, 例如, 三 Glc-人-人和 Glc-人-Glc 将有不同的 Rf。
速度已被广泛用于表征不同的木聚糖结构横跨植物种类8, 以识别糖突变体在拟南芥6,9,10,11, 执行糖检测9, 并为新的 GH 活动特征12,13。我们最近也用它来表征酵母细胞壁甘露聚糖 (Mahboubi 和莫蒂默, 在准备)。在这里, 我们描述了一种植物细胞壁葡甘聚糖结构的表征方法, 根据以前的报告11,14。

<table>
	<tbody>
		<tr>
			<td>Oligosaccaharide Standards</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mannose</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 3458-28-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannobiose</td>
			<td>Megazyme</td>
			<td>CAS: 14417-51-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannotriose</td>
			<td>Megazyme</td>
			<td>CAS: 28173-52-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannotetraose</td>
			<td>Megazyme</td>
			<td>CAS: 51327-76-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannopentaose</td>
			<td>Megazyme</td>
			<td>CAS: 70281-35-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannhexaose</td>
			<td>Megazyme</td>
			<td>CAS: 70281-36-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucomannan (Konjac; Low Viscosity)</td>
			<td>Megazyme</td>
			<td>P-GLCML</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other specialty chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-Aminonaphthalene-1,3,6-trisulfonic acid</td>
			<td>(Molecular probes) Thermo</td>
			<td>A350</td>
			<td></td>
		</tr>
		<tr>
			<td>2-picoline borane</td>
			<td>TCI</td>
			<td>B3018</td>
			<td></td>
		</tr>
		<tr>
			<td>40 % acrylamide/Bis-acrylamide (29:1 acrylamide:Bis)</td>
			<td>Bio-rad</td>
			<td>1610146</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Specialty Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gel casting kit</td>
			<td>Hoefer</td>
			<td>SE660 kit</td>
			<td>18x24 cm glass plates, 0.75 mm spacers</td>
		</tr>
		<tr>
			<td>Cooling recirculating water bath&#160;</td>
			<td>Hoefer</td>
			<td>RCB20-plus 115v</td>
			<td>Needs to be able to maintain temperature at ~10 C</td>
		</tr>
		<tr>
			<td>G:Box Chemi XRQ Imaging System</td>
			<td>Syngene</td>
			<td>05-GBOX-CHEMI-XRQ</td>
			<td>Order with filters appropriate to fluorphore, and transilluminator should be fitted with long-wave UV light bulbs</td>
		</tr>
		<tr>
			<td>High Voltage Power Pack</td>
			<td>Thermo</td>
			<td>EC1000XL</td>
			<td>1000V&#160;</td>
		</tr>
		<tr>
			<td>Vacuum centrifuge(Speedvac)</td>
			<td>Savant</td>
			<td>SPD131</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel electrophoresis system</td>
			<td>Hoefer</td>
			<td>SE660</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycosyl hydrolases</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>These can be obtained from companies e.g. Megazyme (https://www.megazyme.com/) or Prozomix (http://www.prozomix.com/), or can be expressed in house by requesting the plasmid from the relevant research group.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Lab supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge tube ( Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning)</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Centrifuge tube ( Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning)</td>
			<td>VWR</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imaging software ( Genesys)</td>
			<td>Syngene</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

structural characterization of mannan cell wall polysaccharides in plants using pace

植物细胞壁多糖是众所周知的难以分析, 大多数方法需要昂贵的设备, 熟练的操作员, 和大量的纯化材料。在这里, 我们描述了一种获取详细的多糖结构信息的简单方法, 包括结构异构体的解析。用凝胶电泳 (速度) 对多糖进行分析, 用糖水解对植物细胞壁材料进行水解, 其特异性为多糖 (如: mannanases 甘露聚糖)。大格式的聚丙烯酰胺凝胶, 然后用于分离释放的低聚糖, 已被荧光标记。凝胶可以用改良的凝胶体成像系统来可视化 (参见材料表)。由此产生的寡糖指纹可以定性地进行比较, 也可与复制定量地进行对比。可以使用附加的糖水解 (例如, mannosidases 和 galactosidases) 建立链接和分支信息。本协议描述了一种分析葡甘聚糖结构的方法, 可用于任何具有特征糖水解的多糖。另外, 它可以用来表征新的糖水解使用定义的多糖基质。

在这里, 我们展示了一个例子速度凝胶运行的每一个协议, 连同说明, 以协助数据解释和故障排除, 这是后面的一个成功的速度凝胶解释的一般指南13,16。一个代表性的凝胶的细胞壁甘露聚糖含量的标准速度测定显示在图 2, 并描述车道的车道。
标准 车道 1, 2 和3显示商业被购买的低聚糖的梯子 (人1-人6) 在 5 (S1), 10 (S2) 和 50 (S3) pmol 集中。当收到一个新的大量的商业低聚糖, 这是很重要的检查纯度的凝胶。许多寡糖, 特别是 tetrasacccharides 和更长的, 可以有显著的污染与寡糖的其他程度的聚合 (DPs), 如图所示的人6 (标记为 *)。当量化凝胶时, 重要的是要有至少3浓度的标准, 因为它是必要的计算标准曲线。标准曲线的计算也有助于确保衍生化反应基本完成。已知浓度的标准允许对凝胶进行比较, 并且对分离质量和衍生化质量有很好的控制作用。
积极控制 4车道显示了魔芋葡甘聚糖的甘露消化。魔芋葡甘聚糖是可利用的商业, 虽然它没有相同的结构, 例如, 在拟南芥中发现, 它服务两个目的。首先, 它是一种重要的酶消化控制。研究员应该建立什么一个商业基体消化与他们的全球统一制度的选择看起来像当消化到完成 (即,一个巨大的过剩生长激素被添加到反应)。如果在未来的实验中模式改变, 这可能表明要么失去活动或污染的 GH 股票。其次, 在酶和缓冲盐存在的情况下, 它作为衍生化反应的控制。如果标准看起来不错, 但阳性控制较差, 那么这可能表明水解反应的一个组分抑制了衍生。
野生型拟南芥茎空气 + 甘露鸡尾酒 (GH5 + GH26) 车道5显示了一个重量拟南芥茎的速度指纹 (与参考11,14比较)。
csla9拟南芥茎空气 + 甘露鸡尾酒 (GH5 + GH26) 6车道显示的速度指纹的茎从一个拟南芥植物缺乏主要茎甘露聚糖合酶11。比较重量和阴性对照指纹。虽然甘露聚糖的多数在这个植物缺席, 少量保持, 如证明由减少的带强度为所有甘露获得的寡糖类这由另外的甘露酶 (CSLA2, 3)11合成。
阴性控制-仅酵素 8车道显示了胶凝体上的带, 不是从甘露鸡尾酒中的污染物中提取的, 应该从分析中排除 (标有 *)。
负控制-仅限空气 9车道显示胶凝体上的带不具体, 应排除在分析 (标有 *)。
负控制-csla9仅限空气 10车道显示胶凝体上的带不具体, 应排除在分析 (标有 *)。
松树木空气 + 甘露鸡尾酒 (GH5 + GH26) 7车道显示了松树木材的步伐指纹, 其中包含 galactoglucomannan。这显然有不同的模式释放寡糖, 以拟南芥, 由于其不同的结构。
负控制-仅松木空气 11车道显示胶凝体上的带不具体, 应排除在分析 (标有 *)。
解释速度凝胶是相对简单的, 但需要了解以下几点。对于强健的数据, 建议在至少3独立生长的生物复制上执行速度, 建议在每个生物复制上至少执行2技术复制。所描述的控制是解释的关键, 因为非特定的波段需要被排除。我们还建议在复制凝胶上按不同的顺序加载样品, 以排除 transilluminator 不均匀照明所产生的效果。准确的解释需要分析不饱和的波段。由于一些寡糖指纹可能含有非常高和低丰度的多糖, 我们建议分析同一凝胶的多重暴露。这些标准可以用于不同图像之间的规范化。
对于某些类型的实验, 它可能是可取的量化量的多糖在空气中的准备。虽然可以从速度凝胶中量化, 但它需要了解全球统一制度发布的每种寡糖的确切分子身份, 以及它位于佩斯凝胶上的位置。这是目前尚不完全已知的甘露聚糖, 但它已确定为其他多糖如木聚糖3。这些标准确实提供了一种在凝胶之间进行正常化的方法, 即使在不进行定量的情况下也是如此, 因此将有助于进行任何定性或半定量分析。
可以使用全球统一制度的鸡尾酒来确定单个寡糖的结构. 连续的进一步全球统一制度可以揭示有关释放的寡糖的联系和替代的细节 (例如,添加β-14酶在还原端的作用将揭示混合物中的寡糖含有该特征)。可用酶包括 galactosidases、glucuronidases 和 glucosidases (参见 CAZy 数据库16以获取更多信息);见霍格, D. et al.17作为示例。
上面的协议可以用来描述未知的全球统一制度的活动。在这种情况下, 应使用定义的生物量, 如拟南芥茎或魔芋葡甘聚糖, 以筛选未知的全球统一制度13。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56424/56424fig1.jpg"> 
图 1: 这表明了对寡糖与蚂蚁进行荧光衍生的方案.已从 Goubet2中修改。<a href="//ecsource.jov href=""></a href="//ecsource.jov>e.com/files/ftp_upload/56424/56424fig1large.jpg "目标 =" _blank ">> 请点击这里查看更大版本的这个数字。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56424/56424fig2.jpg"> 
图 2:代表速度凝胶显示的甘露聚糖指纹从拟南芥, 松树和拟南芥突变体 (csla9), 这已经损害了甘露聚生物合成.AlR 用甘露鸡尾酒水解, 释放的寡糖衍生荧光。以大小、形状和电荷为基础, 用凝胶电泳分离低聚糖。manno-寡糖 (Man1-6) 的阶梯可以帮助识别释放的寡糖的相对迁移, 而从魔芋中提取的纯化甘露聚糖类的水解是一种阳性对照。* = 非特异带。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56424/56424fig2large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE at JoVE.com

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

介绍了以甘露聚糖为例, 用凝胶电泳 (佩斯) 对多糖进行结构分析的协议。

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

structural-characterization-mannan-cell-wall-polysaccharides-plants

Research

JoVE Journal

Biochemistry

9.5K 次观看.  Joint BioEnergy Institute. 介绍了以甘露聚糖为例, 用凝胶电泳 (佩斯) 对多糖进行结构分析的协议。

视频: Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

LERLIC-MS / MS进行深入表征和谷氨酰胺的定量和天冬酰胺脱酰胺在鸟枪蛋白质组学

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

科研

生物化学

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

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

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 ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角度 x 射线散射溶液中大分子的结构研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...