Name: determination of the glycogen content in cyanobacteria
Uploaded: 2017-07-17T15:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Determination of the Glycogen Content in Cyanobacteria at JoVE.com

作者承认北欧能源研究（AquaFEED，第24号项目），丹麦Innovationfonden（Pant Power，项目编号12-131844）和Villum Fonden（项目编号13363）

准备<ol><li> 蓝藻培养 <ol><li>生长集胞藻PCC 6803在30℃下在液体BG11培养基8中 ，具有补充有1％（v / v）CO 2的恒定空气供应。在光合光子通量密度为50μmol/ m 2 / s的条件下用光照亮培养物。 </li><li>生长聚球藻在液体A +介质23 （也可以使用BG11培养基）中的PCC 7002，补充有1％（v / v）CO 2的恒定空气供应。温度应为37°C。在光合光子通量密度为150μmol/ m 2 / s的光照下连续培养培养物。 </li><li>使用光路径为1厘米的比色皿测量培养物在730nm处的光密度（OD）。如果OD值高于0.8，则进行适当的稀释以获得与th成比例的OD测量值e细胞浓度。 注意:以下提出的方案适用于液体培养，细胞密度对应于OD 730nm值为2或更高。当需要指数生长期的培养物（其通常具有低于1nm的OD 730nm值）时，通过离心浓缩细胞密度并将其重新悬浮在缓冲液或培养基中以达到OD 730nm值为2或更高。 </li></ol></li><li> 缓冲液和试剂 <ol><li>在pH 8下制备50mM Tris-HCl缓冲液。 </li><li>在pH 5下制备50mM乙酸钠缓冲液。 </li><li>在50mM乙酸钠缓冲液（pH 5）中制备8U / mL淀粉葡糖苷酶的储备溶液。 </li><li>在50mM乙酸钠缓冲液（pH 5）中制备2U / mLα-淀粉酶的储备溶液。 </li><li>使用蒸馏水，浓度范围为0至100μg/ mL的葡萄糖标准溶液。 </li><li>从D-葡萄糖测定试剂盒（GODPOD Format）制备GOD-POD试剂，f遵循制造商的指示。 </li></ol></li></ol> 2.细胞干重测定（可选） <ol><li>将2mL培养物或细胞再悬浮液（参见步骤1.1）转移到2.0mL管中，并以6,000xg和4℃离心5分钟。丢弃上清液。 </li><li>将沉淀重悬于1mL水中，并以6,000xg和4℃离心5分钟。弃去上清液，将细胞沉淀重新悬浮于0.5mL水中。 </li><li>将悬浮液转移到预先称重的铝托盘上。将托盘转移到105℃的干燥箱中进行过夜干燥（约18小时）。 关键:请务必用镊子处理托盘，以避免手指中的物料转移。在相同条件下干燥一个空的预先重量的铝托盘，以确定干燥期间托盘的任何重量损失。 </li><li>干燥后，从烤箱中取出托盘，使其在环境条件下平衡称重5分钟，准确度为0.0001克。 注意:该值可用于在细胞干重的基础上标准化糖原含量。 </li></ol> 3.蓝细菌细胞裂解<ol><li>将1 mL培养物或细胞再悬浮液（见步骤1.1）转移至1.5 mL试管中，并以6,000 xg和4℃离心10分钟。丢弃上清液。 </li><li>将沉淀重悬于1mL 50mM Tris-HCl，pH8中，并以6,000xg和4℃离心10分钟。弃去上清液，并将细胞沉淀重悬于1mL Tris-HCl缓冲液中。重复该过程。 </li><li>在500μL50mM Tris-HCl缓冲液（pH8）中彻底重悬细胞沉淀。 关键:重新悬浮沉淀，以获得有效的细胞裂解。保持重新悬浮在冰中。 </li><li> 通过执行30次超声波循环，在4℃下重悬细胞，每个循环由20kHz的频率组成，最大振幅为20kHz，其次是90秒。 注意:这种方法可以有效地溶解聚球藻PCC 7002。 <ol><li>或者，按照制造商的说明，将细胞再悬浮转移到螺旋盖管并将氧化锆珠加入管中。将管置于组织匀浆器中，通过进行2次打浆循环，在4℃下裂解细胞，每个循环由5分钟组成，频率设定为5分钟。 注意:这种方法可以有效地溶解集胞藻（Synechocystis sp。 PCC 6803和聚球藻PCC 7002。 </li></ol></li><li>将含有裂解物的管在6,000 xg和4℃下离心10分钟。 注意:颗粒应主要由大的细胞碎片组成。如果有大量不间断的细胞，请重复步骤3.4。将上清液保存在冰上。 </li><li>使用商业化的BCA蛋白测定试剂盒确定蛋白质浓度。 注意:该值可用于标准化糖原含量在蛋白质含量的基础上。 </li></ol> 4.糖原沉淀<ol><li>通过将900μL96％（v / v）乙醇和100μL来自步骤3.5的上清液在1.5mL螺旋盖管中混合，从细胞裂解物中除去叶绿素a 。关闭盖子后，使用标准实验室加热块将管子在90℃加热10分钟。 </li><li>将管在冰上孵育30分钟。 </li><li>以20,000 xg和4℃离心管30分钟，小心取出上清液;丸粒含有糖原。在空气中轻轻干燥沉淀以除去多余的乙醇。 关键:必须避免颗粒过度干燥，否则在步骤5.1中溶解变得困难。 </li><li>可选:测量在步骤4.3中获得的上清液在664nm处的吸光度，以确定叶绿素a含量。吸收系数为84.6 L / g / cm 24 。 注意:该值可用于normaliz糖原含量。 </li></ol>酶水解和糖原测定<ol><li>将在步骤4.3中获得的颗粒溶解在100μL50mM乙酸钠（pH 5）中，加入50μL8U / mL淀粉葡糖苷酶和50μL2U / mLα-淀粉酶。使用涡流将这些材料充分混合。 注意:不推荐通过移液进行混合，因为糖原颗粒是粘稠的。 </li><li>将混合物在60℃下在加热块上孵育2小时，以使糖原消化成葡萄糖分子。 </li><li>将样品以10,000 xg离心5分钟，并将上清液转移到新的1.5 mL管中。 </li></ol> 6.使用GOD-POD试剂测定总葡萄糖含量<ol><li>使用GOD-POD试剂测量在步骤5.3中获得的上清液中的葡萄糖浓度。将100μL上清液从步骤5.3转移到96孔板中的孔中。作为阴性对照，使用100μL的50mM乙酸钠，pH5。为了产生校准曲线，还测量葡萄糖标准溶液。 </li><li>向每个样品加入150μLGOD-POD试剂，并通过移液快速混合。 </li><li>在25℃静置30分钟。使用平板读数器记录510nm处的吸光度值。 </li><li>使用从葡萄糖标准品获得的校准曲线计算葡萄糖浓度。 注意:细胞裂解物中的糖原浓度以葡萄糖浓度（μg/ mL）表示。 </li></ol>

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作者没有什么可以披露的。

方案中的关键步骤是糖原沉淀和再悬浮。在乙醇沉淀后离心后，糖原形成半透明的颗粒，其松散地粘附到微量离心管的壁上。因此，当除去上清液时，需要特别注意不要去除颗粒。糖原颗粒是粘性的，并且如果干燥，则溶解变得困难。注意，糖原颗粒的完全溶解是重要的，因为不完全溶解将导致低效的酶消化，因此将在技术重复之间产生大的变化。在通过涡旋溶解之前应用超声处理可以有助于该?...

蓝细菌积累糖原作为通过光合作用固定在光中的来自CO 2的碳的主要碳水化合物储存。糖原是由线性α-1,4-连接葡聚糖组成的聚糖，其具有由α-1,6-连接的葡糖基键连接的分支。蓝细菌中的糖原生物合成开始于葡萄糖-6-磷酸转化为ADP-葡萄糖，通过磷酸果糖转移酶和ADP-葡萄糖焦磷酸化酶的顺序作用。 ADP-葡萄糖中的葡萄糖部分通过一种或多种糖原合成酶（GlgA）转移到糖原的α-1,4-葡聚糖骨架的非还原末端。随后，分支酶引入α-1,6-连接的葡糖基键，其进一步延伸以产生糖原颗粒。在黑暗中，糖原被糖原磷酸化酶，糖原脱支酶，α-葡聚糖转移酶和麦芽糊精磷酸化酶分解成磷酸化葡萄糖和游离葡萄糖。这些feed intø分解代谢途径，包括氧化戊糖磷酸途径，Embden-迈耶霍夫-Parnas途径（糖酵解），和ED途径1，2，3，4。 蓝藻中的糖原代谢近年来引起越来越多的兴趣，因为蓝藻潜力发展成由阳光驱动的微生物细胞工厂生产化学品和燃料。由于糖原是这些细菌中最大的柔性碳库，因此可以改变糖原代谢以增加产品的产量。一个例子是蓝细菌聚球藻PCC 7002已被基因工程化生产甘露醇;糖原合成的遗传破坏增加甘露醇产量3倍5 。另一个例子是从加载糖原的野猪生产生物乙醇ype Synechococcus sp。 PCC 7002 6 。野生型细胞的糖原含量可高达细胞的干重的60％的氮饥饿6中。 我们对糖原代谢和调节的理解近年来也有所扩大。虽然糖原已知在光​​中积聚并在黑暗中分解代谢，但在Diel循环中糖原代谢的详细动力学最近才在Synechocystis sp中显现。 PCC 6803 7 。此外，已经鉴定了几种影响糖原积累的基因。一个显着的例子是发现推定的组氨酸激酶PmgA和非编码RNA PmgR1形成调节级联并控制糖原的积累。有趣的是， pmgA和pmgR1缺失突变体与Synechocystis sp。的野生型菌株相比，积累了两倍的糖原。 PCC 68038，9。其他调控元件也已知会影响糖原的积累，包括替代σ因子E和转录因子CyAbrB2 10，11。 由于糖原调节和代谢的兴趣增长，需要一个描述糖原含量测定的详细方案。在文献中使用了几种方法。酸水解然后通过加上一个脉冲电流检测器或在用酸和酚处理光谱法测定高压阴离子交换液相色谱单糖含量的测定被广泛使用的方法来近似糖原含量9，10，12，13。然而，高压阴离子交换液相色谱仪C类仪器是非常昂贵的，并且不区分糖原从来自其它含葡萄糖的糖缀合物，例如蔗糖14，glucosylglycerol 15，和纤维素16，17，18，其已知在一些蓝藻物种积累衍生的葡萄糖。酸 - 酚法可以使用标准实验室设备进行。然而，它使用高毒性试剂，并且不区分来自不同糖缀合物的葡萄糖，也不区分葡萄糖与构成细胞材料的其它单糖，如糖脂，脂多糖和细胞外基质12 。值得注意的是，热酸-苯酚测定经常用于总碳水化合物含量，而不是的葡萄糖含量12的具体确定的确定。酶法通过α-淀粉葡萄糖苷酶将糖原溶解成葡萄糖，然后通过酶偶联测定法检测葡萄糖，产生对来自糖原的葡萄糖高度敏感和特异性的比色读数。特异性可以从细胞裂解物糖原的优先沉淀进一步用乙醇5，8，19来增强。 在这里，我们描述了两个最广泛研究的蓝藻种类Synechocystis sp。的糖基含量的基于酶的测定的详细方案。 PCC 6803和聚球藻PCC 7002，在野生型和突变株中。为了确保有效水解，α淀粉酶和α-淀粉葡萄糖苷酶的混合物被用于8。内效α-淀粉酶将各种葡聚糖中的α-1,4-键水解成糊精，进一步水解为o葡萄糖通过外效α-淀粉葡萄糖苷酶20 。这些酶的协同效应是众所周知的，并且这些酶通常用于选择性水解淀粉，其是α-连接葡聚糖样糖原，而不影响植物生物质21中的其它糖缀合物，例如纤维素。在葡萄糖氧化酶（其催化氧还原为过氧化氢）和葡萄糖氧化成内酯和过氧化物酶（其由过氧化氢产生粉红色醌亚胺染料）组成的酶偶联测定后，定量检测释放的葡萄糖，酚类化合物和4-氨基安替比林22 。

<table><tbody><tr><td>QSonica Sonicators Q700</td><td>Qsonica, LLC</td><td>NA</td><td>QSonica</td></tr><tr><td>SpectraMax 190 Microplate Reader&amp;nbsp;</td><td>Molecular Devices</td><td>NA</td><td>Eliza plate reader</td></tr><tr><td>Bullet Blender Storm</td><td>Next Advance</td><td>BBY24M-CE</td><td>Beads beater</td></tr><tr><td>Ultrospec 3100 pro UV/Visible Spectrophotometer</td><td>Amersham Biosciences</td><td>NA</td><td>Spectrophotometer</td></tr><tr><td>Tris&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1503</td><td>Buffer</td></tr><tr><td>HCl</td><td>Merck</td><td>1-00317</td><td>pH adjutment</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>32319</td><td>Buffer</td></tr><tr><td>Amyloglycosidase (Rhizopus sp.)</td><td>Megazyme</td><td>E-AMGPU</td><td>Enzyme for glycogen depolymerization</td></tr><tr><td>&amp;alpha;-Amylase, thermostable (Bacillus licheniformis)</td><td>Sigma-Aldrich</td><td>A3176</td><td>Enzyme for glycogen depolymerization</td></tr><tr><td>D-Glucose</td><td>Merch</td><td>8337</td><td>Standard for the glucose assay</td></tr><tr><td>Pierce BCA Protein assay kit&amp;nbsp;</td><td>Thermo Fisher scientific</td><td>23225</td><td>For determination of protein concentrations</td></tr><tr><td>Aluminum drying trays, disposable</td><td>VWR</td><td>611-1362</td><td>For determination of cell dry weights</td></tr><tr><td>D-Glucose assay kit (GODPOD format)</td><td>Megazyme</td><td>K-GLUC</td><td>For determination of glucose concentrations</td></tr><tr><td>Zirconium oxide breads, 0.15 mm</td><td>Next Advance</td><td>ZrOB015</td><td>Beads for cell lysis in a Bullet Blendar Storm</td></tr><tr><td>RINO tubes</td><td>Next Advance</td><td>NA</td><td>Tubes for cell lysis in a Bullet Blendar Storm</td></tr></tbody></table>

determination of the glycogen content in cyanobacteria

蓝藻在光合作用过程中积累糖原作为主要的细胞内碳和能量储存。研究的最新进展突出表明糖原代谢的复杂机制，包括生物合成和分解代谢的diel循环，氧化还原调节和非编码RNA的参与。同时，正在努力将碳从糖原转移到转基因工程蓝细菌中的所需产品，以提高产品产量。用几种方法测定蓝细菌中的糖原含量，具有不同的准确性和技术复杂性。在这里，我们提供了一个详细的协议，可以确定可以在标准生命科学实验室进行的蓝细菌中的糖原含量。该方案需要从细胞裂解液中糖原的选择性沉淀和糖原的酶促解聚以产生葡萄糖单体，其由葡萄糖酪氨酸过氧化物酶（GOD-POD）酶偶联测定。该方法已应用于集胞藻（Synechocystis sp。 PCC 6803和聚球藻PCC 7002，两种广泛应用于代谢工程的蓝藻种类。此外，该方法成功地显示了调节元件或糖原生物合成基因中的野生型和突变体之间的糖原含量的差异。

10 mL野生型集胞藻PCC 6803在光自养条件下生长，直到OD 730nm值达到约0.8。收集细胞并重悬于50mM Tris-HCl，pH8中。将OD 730nm值调节至2-3。按照上述方案分析糖原含量。每个OD 730nm的糖原含量为13±1.8μg/ mL / OD 730nm （ N = 12）。糖蛋白含量相对于蛋白质含量为0.24±0.03μg/μg（ N = 12），相对于叶绿素a含量的糖原含量为5.7±0.6μ...

Watch this Scientific Journal Video about Determination of the Glycogen Content in Cyanobacteria at JoVE.com

Determination of the Glycogen Content in Cyanobacteria

在这里，我们提出了一个可靠和容易的测定来测量蓝细菌细胞中的糖原含量。该过程需要沉淀，可选择解聚和葡萄糖残留的检测。该方法适用于野生型和遗传工程菌株，可促进蓝细菌的代谢工程。

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

determination-of-the-glycogen-content-in-cyanobacteria

Research

JoVE Journal

Biochemistry

12.9K 次观看.  Carlsberg Research Laboratory. 在这里，我们提出了一个可靠和容易的测定来测量蓝细菌细胞中的糖原含量。该过程需要沉淀，可选择解聚和葡萄糖残留的检测。该方法适用于野生型和遗传工程菌株，可促进蓝细菌的代谢工程。 

视频: Determination of the Glycogen Content in Cyanobacteria

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

分光光度法测定蓝藻Synechocystis中的蛋白含量

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

科研

生物化学

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

免疫与感染

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

用于研究真核糖原代谢的分光光度法

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

肝糖原分子的提取用于糖原结构测定

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

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 ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

生物学

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

蓝藻中完整藻胆体的分离与表征

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

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

Source: Wilson, W. A., <a href="https://app.jove.com/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
In this video, we describe a qualitative spectrophotometric assay to determine the extent of branching in a glycogen sample with an uncharacterized structure. Comparing the absorbance maxima of the uncharacterized sample with that of polysaccharides with known branched structures indicates the presence of branching in the uncharacterized sample.

糖原支化测定法，用于测量糖原支化的程度

Source: Wilson, W. A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
In this video, we describe a ...