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过程。 关于归一化方法的选择，细胞干重是广泛使用的参考。比蛋白质和叶绿素的测定更费力，需要更多的细胞生物量a 。总蛋白质含量提供了对细胞生物质的替代参考，并且可以如本方案所述在小规模上测定。每干细胞生物量的总蛋白质含量通常在40％和50％之间的范围内，但确切值取决于生长条件25，28。可以容易地测定叶绿素a的含量，可以作为样品中存在的细胞量的代表。然而，众所周知，每个细胞的叶绿素a的量根据生长条件而显着变化，特别是响应于不断变化的营养物浓度和光强度29 。 所提出的技术相对于其他方法的显着优点之一是其具有高选择性。热酸和苯酚方案和单糖组成分析如下酸水解是比较简单的方法和在以前的研究中9，10，12，13已经被使用。然而，这些方法可以高估糖原含量，因为非糖原葡萄糖和额外的糖可以有助于测量，这取决于碳水化合物检测技术12 。所描述的技术选择性地检测糖原中的葡萄糖。由于α-淀粉酶和α-淀粉葡萄糖苷酶不会水解存在于纤维素中的β-连接的葡糖基键，所以它也可以区别葡萄糖与纤维素。在以前的研究中，用α-淀粉葡糖苷酶7，27单独运行的糖原的酶水解。如本协议所述，将内效α-淀粉酶与外作用的α-淀粉葡萄糖苷酶一起包含在内，可以确保t他有效地水解糖原。通常将类似的选择性水解应用于植物生物量的处理，其中α-淀粉酶α-淀粉葡萄糖苷酶的组合用于水解淀粉而不影响其它含葡萄糖的多糖如纤维素21 。 该技术的主要限制是，由于使用微量离心管单独处理单个样品，因此该方法是低通量的。糖原沉淀步骤是防止更高产量的主要因素。作为高通量程序的实施将需要使用深孔板和能够以高速离心（20,000× g ）的能力。虽然可以以必要速度离心96孔板的离心机，但大多数深井板不能容忍大于6,000 x g的力 。因此，需要仔细选择材料以适应高分辨率的协议h通量分析。

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 border="0" cellpadding="0" cellspacing="0" width="784">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">QSonica Sonicators Q700</td>
			<td style="width:219px;">Qsonica, LLC</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;">QSonica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax 190 Microplate Reader&#160;</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:119px;">NA</td>
			<td>Eliza plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bullet Blender Storm</td>
			<td style="width:219px;">Next Advance</td>
			<td style="width:119px;">BBY24M-CE</td>
			<td>Beads beater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrospec 3100 pro UV/Visible Spectrophotometer</td>
			<td style="width:219px;">Amersham Biosciences</td>
			<td style="width:119px;">NA</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">HCl</td>
			<td style="width:219px;">Merck</td>
			<td style="width:119px;">1-00317</td>
			<td>pH adjutment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">32319</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amyloglycosidase (Rhizopus sp.)</td>
			<td style="width:219px;">Megazyme</td>
			<td style="width:119px;">E-AMGPU</td>
			<td>Enzyme for glycogen depolymerization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Amylase, thermostable (Bacillus licheniformis)</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3176</td>
			<td>Enzyme for glycogen depolymerization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:219px;">Merch</td>
			<td style="width:119px;">8337</td>
			<td>Standard for the glucose assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce BCA Protein assay kit&#160;</td>
			<td>Thermo Fisher scientific</td>
			<td style="width:119px;">23225</td>
			<td>For determination of protein concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Aluminum drying trays, disposable</td>
			<td style="width:219px;">VWR</td>
			<td style="width:119px;">611-1362</td>
			<td>For determination of cell dry weights</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">D-Glucose assay kit (GODPOD format)</td>
			<td style="width:219px;">Megazyme</td>
			<td style="width:119px;">K-GLUC</td>
			<td>For determination of glucose concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Zirconium oxide breads, 0.15 mm</td>
			<td style="width:219px;">Next Advance</td>
			<td style="width:119px;">ZrOB015</td>
			<td>Beads for cell lysis in a Bullet Blendar Storm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">RINO tubes</td>
			<td style="width:219px;">Next Advance</td>
			<td style="width:119px;">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±0.6μg/μg（ N = 12）。由于可用的材料量少，所以省略了细胞干重的测量。鉴于在相当条件下培养的蓝细菌中的蛋白质含量约为细胞干重25 ％的50％/ sup&gt;，糖原含量估计为细胞干重的12％，这与以前的研究26一致。 GOD-POD测定的检测限显示在10至100μg/ mL的葡萄糖浓度范围内，葡萄糖浓度与510nm处吸光度之间呈线性相关，对应于510nm处的0.08和0.7的吸光度值。最小限制可能是由于仪器检测限制。当葡萄糖浓度高于150μg/ mL时，形成深绿色沉淀，导致吸光度读数大幅度变化。关于电池材料的使用量，我们经常获得可再现的糖原含量时细胞再悬浮的OD 730nm处值为之前的细胞裂解2个10.细胞再悬浮之间为1的OD 730nm处的值或低于给人们带来接近信号最小检测限t，导致高度可变的结果。 OD 730nm值高于20的细胞再悬浮不适合，因为细胞裂解不完全，需要延长裂解或稀释。 图1显示了Synechocystis sp。中糖原含量的代表性结果。 PCC 6803野生型和两株突变菌株（ ΔpmgA和ΔpmgR1 ）。使用以指数生长期生长的细胞。糖原含量首先通过总蛋白质含量进行归一化，随后以相对于野生型的值表示。结果表明，突变菌株的糖原含量比野生型菌株高2倍以上。 接下来，在聚球藻属菌株中分析糖原含量。 PCC 7002设计用于生产甘露醇5<html> 。第一应变（GLG +）包含编码两个功能糖原合酶与野生型glgA1和glgA2基因，而第二应变（GLG - ）缺乏这些基因5的功能性拷贝。然后在两个菌株中测定糖原和甘露醇含量（ 图2 ）。该结果表明，GLG -缺乏糖原的可检测水平，而它产生比GLG +应变更多甘露醇。这表明通过光合作用合成的碳水化合物被转移到缺乏合成糖原的能力的突变株中的甘露醇中。培养物的OD 730nm值约为10，为细胞干重分析提供了足够的细胞材料。使用细胞干重使糖原含量标准化。 8 / 56068fig1.jpg"/&gt; 图1: 在不同的行集胞藻的测得的糖原含量。 PCC在WT和在两个突变体（ΔpmgA9和ΔpmgR18）6803相对糖原内容被示出。糖原水平用总蛋白质含量标准化。显示三种生物重复的方法，误差棒代表标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56068/56068fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56068/56068fig2.jpg" /> 图2: 遗传操纵的聚球藻属中的糖原和甘露醇生产PCC 7002 5 。 GLG &lt;sup&gt; +，该菌株合成甘露醇和糖原。 Glg - ，合成甘露醇但不含糖原的菌株。所描绘的值是三个生物重复的平均值，误差棒代表标准偏差。 CDW:细胞干重，ND:未检出。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56068/56068fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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

Nanyang Technological University

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

高亲和力抗体 - 抗原的结合动力学的测定，使用四个生物传感器平台

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in lipid droplets (LDs). LD index is obtained without lipid extraction. It allows measuring the LDs content in high-throughput experiments under different conditions such as growth in rich or nitrogen depleted media. Albeit the method was described for the first time to study the lipid droplet metabolism in Saccharomyces cerevisiae, it was successfully applied to the basidiomycete Ustilago maydis. Interestingly, and because LDs are organelles phylogenetically conserved in eukaryotic cells, the method can be applied to a large variety of cells, from yeast to mammalian cells. The LD index is based on the liquid fluorescence recovery assay (LFR) of the BODIPY 493/503 under quenching conditions, by the addition of cells fixed with formaldehyde. Potassium iodine is used as a fluorescence quencher. The ratio between the fluorescence and the optical density slopes is named LD index. Slopes are calculated from the straight lines obtained when BODIPY fluorescence and optical density at 600 nm (OD600) are plotted against sample addition. Optimal data quality is reflected by correlation coefficients equal or above 0.9 (r &#8805; 0.9). Multiple samples can be read simultaneously as it can be implemented in a microplate. Since BODIPY 493/503 is a lipophilic fluorescent dye that partitions into the lipid droplets, it can be used in many types of cells that accumulate LDs.

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

Here, we describe a protocol to obtain the lipid droplet index (LD index) to study the dynamics of triacylglycerols in cells cultured in high-throughput experiments. The LD index assay is an easy and reliable method that uses BODIPY 493/503. This assay does not need dispendious lipid extraction or microscopy analysis.

真菌病原体液体荧光恢复法测定脂质指数黑穗病玉米

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in ...

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 ...

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 ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

实现蛋白质快速光化学氧化的实时补偿，用于确定蛋白质地形变化

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

使用硫二甘酸（TGA）估计植物生物质木质素含量

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...