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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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>&lt;strong&gt;Oligosaccaharide Standards&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other specialty chemicals&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Specialty Equipment&lt;/strong&gt;</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&amp;nbsp;</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&amp;nbsp;</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>&lt;strong&gt;Glycosyl hydrolases&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Lab supplies&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</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/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

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

科研

生物化学

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

化学

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

生物学

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

发育生物学

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached to each other by &#945;-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, &#946;-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis FACE Method

In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen.

使用荧光团辅助碳水化合物电泳（FACE）方法测定糖原的葡聚糖链长分布

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays 

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

使用微阵列聚合物分析阐明聚糖结构和存在

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

作者聚焦：MAPP 实验方案 – 推进游离寡糖分析 

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

用于高通量聚糖分析的微阵列聚合物分析 （MAPP）

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...

植物甘露聚糖细胞壁多糖的结构表征

摘要

探索更多视频

此视频中的章节

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

使用荧光团辅助碳水化合物电泳（FACE）方法测定糖原的葡聚糖链长分布

胞外多糖的OLIGO质谱分析（OLIMP）

糖基谱研究植物细胞壁聚合物，用微阵列

结晶纤维素积累高分辨率的量化

用于高通量聚糖分析的微阵列聚合物分析（MAPP）

植物甘露聚糖细胞壁多糖的结构表征

摘要

探索更多视频

此视频中的章节

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

使用荧光团辅助碳水化合物电泳（FACE）方法测定糖原的葡聚糖链长分布

胞外多糖的OLIGO质谱分析（OLIMP）

糖基谱研究植物细胞壁聚合物，用微阵列

结晶纤维素积累高分辨率的量化

用于高通量聚糖分析的微阵列聚合物分析 （MAPP）

用于高通量聚糖分析的微阵列聚合物分析（MAPP）