Name: analysis of athird11 intrinsic disorder and binding towards metal ions by capillary gel electrophoresis and affinity capillary electrophoresis
Uploaded: 2018-08-22T14:30:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis at JoVE.com

我们感谢 Masakuza (日本静冈县大学绿色科技研究院) 提供 AtHIRD11 蛋白质样品。

1. 毛细管电泳仪器的制备
<ol><li>
准备毛细血管
	<ol>
<li>使用玻璃切割机切割一个裸熔融二氧化硅毛细管与聚酰亚胺外涂层和内径为50µm 到33厘米 (为 CGE 实验) 和30厘米 (ACE 实验) 长毛细血管。在玻璃板上进行切割。 注: 为2种不同的仪器开发了2种不同的实验装置。为了在其他仪器上使用它们, 必须进行适当的方法转移, 并且必须将毛细管长度调整到仪器的允许长度。</li>
		<li>使用钢笔标记1厘米宽检测窗口的中间, 距离24.5 厘米, 从毛细管的一端到 CGE 实验, 从毛细管一端的21.5 厘米到 ACE 实验 (有效长度)。</li>
		<li>将聚酰亚胺外部涂层从毛细血管中取出, 用一个焊枪, 在标记前后0.5 厘米处燃烧。同样, 使用焊枪去除1厘米的涂层两端的毛细血管。 注: 有效长度可能略有不同, 因为它依赖于使用的毛细管电泳仪器。</li>
		<li>用乙醇和软组织清洁毛细血管和检测窗的两端。 注: 未涂布毛细血管不太灵活, 可能会断裂。</li>
	</ol>
</li>
	<li>
安装毛细血管
	<ol>
<li>使用靠近插座的检测窗口将毛细管安装到 CE 系统中。 注: 各种 CE 仪器型号对毛细血管有不同的保存系统。有关确切说明, 请参阅所用仪器手册。</li>
	</ol>
</li>
</ol>2. 准备解决方案
<ol><li>为 CGE 分析准备解决方案
	<ol>
<li>在 pH 8.0 上制备三羟甲基氨基甲烷 (三)-SDS 缓冲液 (100 mM/1%w/v)。<ol>
<li>使用呼吸面罩和展位, 在100毫升烧杯中称量1.00 克月桂酸钠 (SDS)。</li>
			<li>在100毫升烧杯中加入1.21 克, 用50毫升的去离子水和磁力搅拌器上的磁力搅拌棒将其溶解。</li>
			<li>将 ph 电极放入溶液中, 用1.0 米 HCl 将 ph 值调整为8.0。</li>
			<li>将溶液填入100毫升的容积烧瓶中, 加入去离子水直至标记。轻轻摇动, 以避免任何泡沫成型。</li>
		</ol>
</li>
		<li>准备0.1 米氢氧化钠溶液。<ol>
<li>在100毫升容积烧瓶中重4.0 克氢氧化钠。用去离子水填满它的标记, 使1米的股票。</li>
			<li>使用10毫升的灯泡吸管将这个溶液的10毫升转移到另一个100毫升的容积瓶中, 用去离子水填满它的标记。</li>
		</ol>
</li>
		<li>准备0.1 米 HCl 溶液。<ol>
<li>用10毫升容积吸管将10毫升的10米 HCl 放入100毫升容积瓶中, 用去离子水填充到标记上。重复此步骤以获取 0.1 M HCl。</li>
		</ol>
</li>
		<li>准备示例解决方案。<ol>
<li>将0.5 毫克的蛋白质称量成1毫升管。添加0.5 毫升的三 SDS 凝胶缓冲液从步骤2.1.1 与微。</li>
			<li>通过轻轻摇动来溶解蛋白质, 以避免任何泡沫的形成和蛋白质的降解。 注: 如果蛋白质不能如所描述的那样溶解, 请使用超声波;避免任何加热的解决方案。</li>
		</ol>
</li>
		<li>把溶液装进瓶子里。<ol>
<li>通过0.22 µm 聚偏氟乙烯 (PVDF) 过滤器过滤每个溶液, 用适当的注射器直接进入瓶子。 注: 对于含凝胶溶液, 由于其高粘度, 背压可能很高。</li>
			<li>总共准备15瓶, 并标记每个瓶子, 以避免任何混淆。<ol>
<li>在 pH 值为8的情况下, 用可商用的月桂酸钠 (SDS) 凝胶缓冲液填充3瓶。 注: 使用商用 SDS 凝胶缓冲液可提供更精确的结果。</li>
				<li>填充1瓶0.1 米氢氧化钠和1瓶与0.1 米 HCl 冲洗。</li>
				<li>用样品溶液填充1瓶。</li>
				<li>用去离子水和4瓶0.1 毫升的去离子水 (浸渍瓶) 填充5瓶。 注: 在每次运行前, 在毛细管冲洗过程中, 浸渍瓶被用作废瓶子。毛细管末端浸在水中, 以冲洗任何凝胶残渣。</li>
			</ol>
</li>
		</ol>
</li>
	</ol></li>
	<li>为 ACE 分析准备解决方案
	<ol>
<li>准备1米氢氧化钠溶液。<ol>
<li>在100毫升容积烧瓶中重4.0 克氢氧化钠。</li>
			<li>用去离子水填满它的标记, 使1米的股票。</li>
		</ol>
</li>
		<li>在0.1 米氢氧化钠溶液中制备0.1 米 ethylendiaminetetraacetic 酸 (EDTA)。<ol>
<li>使用10毫升灯泡吸管在100毫升容积烧瓶中转移10毫升0.1 米氢氧化钠。用去离子水填满它的标记。</li>
			<li>在一条称量船上重2.42 克 EDTA, 在100毫升的0.1 米氢氧化钠中溶解固体化合物。</li>
		</ol>
</li>
		<li>准备三缓冲器 (30 毫米) 溶液。<ol>
<li>添加3.63 克的三, 200 毫升的去离子水, 并在500毫升烧杯搅拌棒。把烧杯放在搅拌板上, 然后打开。</li>
			<li>将 ph 表电极放入烧杯中, 用0.1 米 HCl 将 ph 值调整为7.4。</li>
			<li>用1升容积的烧瓶填充溶液, 用去离子水填满标记。 注: 这一程序必须重复或更大的容积瓶是需要的, 因为超过 1 L 是需要的整个分析。</li>
		</ol>
</li>
		<li>制备乙酰苯胺渗流 (EOF)-标记 (60 µM) 溶液。<ol>
<li>在100毫升容积烧瓶中加入6毫克的乙酰苯胺和100毫升的三个缓冲器。</li>
			<li>将容积瓶放入超声波浴中, 并将其打开30分钟。</li>
		</ol>
</li>
		<li>准备样品 (1 毫克/毫升) 溶液。<ol>
<li>把一个0.5 毫升离心管在一个精确的平衡, 并添加0.3 毫克冻干 AtHIRD11 粉入管。</li>
			<li>使用微在管中添加0.3 毫升30毫米的三缓冲器 (pH 7.4)。仔细摇动管子以溶解蛋白质。</li>
		</ol>
</li>
		<li>准备配体溶液。<ol>
<li>准备 CaCl2 (5 毫米) 库存解决方案。<ol>
<li>取一条称重的船, 把它放在分析天平上, 重36.76 毫克的 CaCl2* H2O。</li>
				<li>使用微, 将 CaCl2* H2O 从称量船上冲洗成50毫升容积烧瓶, 并预先准备好的三缓冲器。</li>
				<li>用三个缓冲器填满容积瓶, 直至标记。</li>
			</ol>
</li>
			<li>准备剩余的库存解决方案 (5 毫米)。<ol>
<li>重复步骤2.2.6.1 使用其他金属盐而不是 CaCl2* H2O [氯化镁2: 23.80 毫克, BaCl2: 26.02 毫克, Sr (NO3)2: 52.91 毫克, MnCl2: 31.46 毫克, SeCl4: 52.91 毫克, CoCl2* 2H2O: 41.47 毫克, CuCl2* 2H2o: 42.62 毫克, ZnCl2: 3.41 毫克, NiCl2* 6H2O: 59.43 毫克]。 注: ZnCl2溶液的浓度为0.5 毫米。由于离子与内毛细管壁之间的强相互作用, 浓度降低了 104。</li>
			</ol>
</li>
			<li>准备500µM CaCl2溶液。<ol>
<li>填充10毫升的 CaCl2库存解决方案, 并把它在100毫升容积瓶。</li>
				<li>用三个缓冲器将容积瓶填满, 然后摇动烧瓶。</li>
			</ol>
</li>
			<li>准备250µM CaCl2溶液。<ol>
<li>填充5毫升的 CaCl2库存解决方案, 并把它在100毫升容积瓶。</li>
				<li>用三个缓冲器将容积瓶填满, 然后摇动烧瓶。</li>
			</ol>
</li>
			<li>对其他库存解决方案重复步骤2.2.6.3 和2.2.6.4。</li>
		</ol>
</li>
		<li>把溶液装进瓶子里。 注意: 每次重复运行需要一组入口和出口瓶。在入口和出口处使用新鲜配体溶液可以减少每次运行后迁移时间的变化。<ol>
<li>用10毫升注射器填充250µM CaCl2溶液。</li>
			<li>在注射器上放置一个0.22 µm PVDF 过滤器, 并通过过滤器推2毫升的溶液, 用注射器, 以丢弃这个过滤的解决方案。</li>
			<li>填充10瓶高达最大允许体积与其余250µM CaCl2溶液的注射器。标记为250 µM CaCl 2溶液入口瓶。</li>
			<li>填充10瓶, 直到他们半填充250µM CaCl2解决方案。标记为250 µM CaCl 2溶液出口瓶。</li>
			<li>重复步骤 2.2.7.1-2.2.7.4 的其他金属含盐溶液。</li>
			<li>使用 30 mM 三缓冲器 (pH 7.4) 溶液代替每对入口和出口瓶的步骤。 注:30 毫摩尔/升三缓冲器 pH 值7.4 用于在运行之间, 以获得的迁移时间数据的蛋白质缺乏金属离子。要忽略 EOF 的变化是必要的。</li>
			<li>使用乙酰苯胺 EOF 标记 (60 µM) 解决方案, 对一小瓶重复上述步骤。另外, 请使用示例 (1 毫克/毫升) 解决方案来重复这些步骤。</li>
		</ol>
</li>
	</ol></li>
</ol>3. 毛细管凝胶电泳分离
注: 根据 Nachbar等以前工作中描述的方法准备分离。4。
<ol><li>
准备分析
	<ol>
<li>
条件毛细管
		<ol>
<li>将恒温器设置为23摄氏度。</li>
			<li>用0.1 米氢氧化钠在2.5 巴上冲洗毛细管10分钟。</li>
			<li>冲洗毛细管5分钟在2.0 酒吧与0.1 米 HCl。</li>
			<li>在2.0 酒吧用去离子水冲洗毛细管2分钟。</li>
		</ol>
</li>
		<li>
填充 SDS 凝胶缓冲液
		<ol>
<li>在2.0 巴上填充毛细管10分钟, 在 8.0 pH 值的商业上可用 SDS 凝胶缓冲剂。</li>
		</ol>
</li>
		<li>
在每次分离运行前冲洗毛细管
		<ol>
<li>用0.1 米氢氧化钠在4.0 巴上冲洗毛细管3分钟。</li>
			<li>冲洗毛细管1分钟在4.0 酒吧与0.1 米 HCl。</li>
			<li>在4.0 酒吧用去离子水冲洗毛细管1分钟。</li>
			<li>用 SDS 凝胶缓冲液在4.0 巴上冲洗毛细管10分钟。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
运行分离
	<ol>
<li>为 CGE 实验 (步骤 2.1.4) 水力注入样品溶液, 在进气口应用0.1 条4分钟。</li>
		<li>应用-16.5 伏和2.0 巴的压力在毛细管两端为25分钟。</li>
	</ol>
</li>
</ol>4. 亲和毛细管电泳分析
注: 根据 Nachbar等先前作品中描述的方法准备分离。4和 Alhazmi等。5。
<ol><li>
条件毛细管
	<ol>
<li>将恒温器设置为23摄氏度。 注: 温度根据发布的协议使用, 并且可以被改变到其他温度4,5。然而, 相互作用取决于温度并且将相应地改变。</li>
		<li>用0.1 米氢氧化钠在2.5 巴上冲洗毛细管40分钟。</li>
		<li>在1.0 酒吧用去离子水冲洗毛细管10分钟。</li>
		<li>用30毫米三缓冲器 (pH 7.4) 在1.0 巴上冲洗毛细管30分钟。</li>
	</ol>
</li>
	<li>
ACE 方法的准备
	<ol>
<li>准备没有配体的测量方法。 注意: 乙酰苯胺没有加入到样品溶液中, 因为它掩盖了2蛋白峰值。<ol>
<li>用0.1 米 EDTA 溶液在2.5 巴上冲洗毛细管1分钟。</li>
			<li>在2.5 酒吧用去离子水冲洗毛细管1分钟。</li>
			<li>在2.5 巴上冲洗毛细管1.5 分钟, 用三个缓冲器进行平衡。</li>
			<li>注射乙酰苯胺溶液 6 s 在0.05 酒吧和改变入口和出口瓶到三个缓冲瓶。</li>
			<li>应用 0.05 bar 2.4 s, 以推动乙酰苯胺溶液从毛细管尖端进一步内。</li>
			<li>应用10.0 伏6分钟, 以 200 nm 的波长检测乙酰苯胺峰值。</li>
			<li>重复步骤4.2.1.1。-4.2.1.6。使用蛋白质样本代替乙酰苯胺溶液, 并检测所有蛋白质峰值。</li>
		</ol>
</li>
		<li>用配体制备测量方法。<ol>
<li>用0.1 米 EDTA 溶液在2.5 巴上冲洗毛细管1分钟。</li>
			<li>在2.5 酒吧用去离子水冲洗毛细管1分钟。</li>
			<li>用配体溶液进行平衡, 在2.5 巴上冲洗毛细管1.5 分钟。</li>
			<li>注射乙酰苯胺溶液 6 s 在0.05 酒吧和改变入口和出口瓶到配体包含缓冲瓶。</li>
			<li>应用 0.05 bar 2.4 s, 以推动乙酰苯胺溶液从毛细管尖端进一步内。</li>
			<li>应用10.0 伏6分钟, 以 200 nm 的波长检测乙酰苯胺峰值。</li>
			<li>重复步骤 4.2.2.1-4.2.2.6 使用蛋白质样本, 而不是乙酰苯胺溶液和检测所有的蛋白质峰值。</li>
		</ol>
</li>
		<li>交替重复步骤4.2.1 和 4.2.2, 以计算各种蛋白质-金属离子相互作用的电荷大小比值的变化 (在代表性结果下描述)。<ol>
<li>每60小时更换一次新的蛋白质溶液。 注意: 在这一点上, 实验可以暂停。这个过程是至少重复10x 为每个配体的浓度, 因为峰值模式变化略有不同运行。如果该解决方案使用的时间超过60小时, 则变体变得太大。</li>
		</ol>
</li>
		<li>
运行方法
		<ol>
<li>运行在步骤4.2.2 下描述的方法, 使用碱性地球配体溶液 (CaCl2, 氯化镁2, BaCl2, SrCl2溶液)。</li>
			<li>继续使用在步骤4.2.2 下描述的方法, 使用 MnCl2、SeCl4、CoCl2、CuCl2、ZnCl2和 NiCl2解决方案而不是碱性地球配体解决方案。 注: 后过渡金属氯化物显示与毛细管更强的相互作用, 不能完全去除。这些交互导致迁移时间从运行转移到运行。因此, 他们被调查后, 其他金属离子。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctionality+of+dehydrins:+An+overview.">The multifunctionality of dehydrins: An overview.</a> Plant Signaling &amp; Behavior. 5 (5), 1-6 (2010).</li><li>Uversky, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dancing+protein+clouds:+the+strange+biology+and+chaotic+physics+of+intrinsically+disordered+proteins.">Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.</a> Journal of Biological Chemistry. 291 (13), 6681-6688 (2016).</li><li>Hara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+the+Arabidopsis+KS-type+dehydrin+protein,+whose+gene+expression+is+constitutively+abundant+rather+than+stress+dependent.">Biochemical characterization of the Arabidopsis KS-type dehydrin protein, whose gene expression is constitutively abundant rather than stress dependent.</a> Acta Physiologia Plantarum. 33 (6), 2103-2116 (2011).</li><li>Nachbar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ion+-+dehydrin+interactions+investigated+by+affinity+capillary+electrophoresis+and+computer+models.">Metal ion - dehydrin interactions investigated by affinity capillary electrophoresis and computer models.</a> Journal of Plant Physiology. 216, 219-228 (2017).</li><li>Alhazmi, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+platform+to+investigate+protein-metal+ion+interactions+by+affinity+capillary+electrophoresis.">A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.</a> Journal of Pharmaceutical and Biomedical Analysis. 107, 311-317 (2015).</li><li>Redweik, S., Xu, Y., W&#228;tzig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise,+fast,+and+flexible+determination+of+protein+interactions+by+affinity+capillary+electrophoresis:+Part+1:+Performance.">Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: Part 1: Performance.</a> ELECTROPHORESIS. 33 (22), 3316-3322 (2012).</li><li>Ghosal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetic+flow+and+dispersion+in+capillary+electrophoresis.">Electrokinetic flow and dispersion in capillary electrophoresis.</a> Annual Review of Fluid Mechanics. 38 (1), 309-338 (2006).</li><li>Xuan, X., Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+study+of+Joule+heating+effects+on+electrokinetic+transportation+in+capillary+electrophoresis.">Analytical study of Joule heating effects on electrokinetic transportation in capillary electrophoresis.</a> Journal of Chromatography A. 1064 (2), 227-237 (2005).</li><li>Oledzka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+tissue+protein+separations+applying+one-+dimensional+gel+electrophoresis+and+capillary+gel+electrophoresis.">Comparative evaluation of tissue protein separations applying one- dimensional gel electrophoresis and capillary gel electrophoresis.</a> The Open Proteomics Journal. 5 (1), 17-21 (2012).</li><li>Alhazmi, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+affinity+capillary+electrophoresis+for+routine+investigations+of+protein-metal+ion+interactions.">Optimization of affinity capillary electrophoresis for routine investigations of protein-metal ion interactions.</a> Journal of Separation Science. 38 (20), 3629-3637 (2015).</li><li>Busch, M. H. A., Carels, L. B., Boelens, H. F. M., Kraak, J. C., Poppe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+methods+for+the+study+of+drug-protein+binding+in+affinity+capillary+electrophoresis.">Comparison of five methods for the study of drug-protein binding in affinity capillary electrophoresis.</a> Journal of Chromatography A. 777 (2), 311-328 (1997).</li><li>Mozafari, M., Balasupramaniam, S., Preu, L., El Deeb, S., Reiter, C. G., W&#228;tzig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+affinity+capillary+electrophoresis+and+computational+models+for+binding+studies+of+heparinoids+with+P-selectin+and+other+proteins.">Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with P-selectin and other proteins.</a> ELECTROPHORESIS. 38 (12), 1560-1571 (2017).</li></ol>

作者没有什么可透露的。

对于所有的 CE 实验, 毛细管的制备是一个关键步骤。由于它是一个小直径的玻璃管, 它可以很容易地打破在涂层被删除时, 它正在处理。将毛细管安装到仪器中应该非常小心。
使用 ACE 方法所涉及的关键步骤主要与实验设置有关。另一个关键步骤是找到正确的样本注入参数。样品的注入量必须足够高的峰值。然而, 重载它将导致一个不太解决的峰值模式与重叠的峰值。在减小注...

植物比其他许多生命形式更依赖于他们的环境。由于植物不能迁移到其他地方, 它们必须适应周围环境的变化 (例如干旱、寒冷和高盐浓度)。因此, 高等植物开发了像 dehydrins 这样的特殊的应力蛋白, 它完成多种任务, 减少与高盐度有关的细胞应力。这些蛋白结合细胞内的水和离子, 通过结合铜2 +离子来减少氧化应激, 并与磷脂和骨架相互作用。此外, 结合锌2 +离子允许这些蛋白质作为转录因子。在磷酸化后, 它们结合钙2 +离子的能力也被报告了1。
这些蛋白质的多功能行为与无疏水性氨基酸残留有关。因此, 它们缺乏肽链内的任何疏水相互作用, 也没有受约束的结构。然而, 由于这些蛋白质缺乏限制性结构, 它们可以在相同的条件下占据不同的构象。因此, 它们可以被描述为结构的集合, 而不是单一的构造。这些性质的蛋白质被称为内在紊乱的蛋白质 (国内流离失所者), 是一个广泛使用的概念, 在应力蛋白和串扰之间的不同途径真核细胞在真核细胞2。
这些与压力有关的国内流离失所者之一是 AtHIRD11。它是拟南芥最干旱表达的国内流离失所者之一。因此, 不同的构象可以用其有效的半径与电荷比分开, 毛细管电泳 (CE) 已被用于进一步的研究。先前的 ACE 实验证明了 AtHIRD11 和过渡金属离子的相互作用, 如铜2 +-锌2 +-Co2 +和镍2 +离子。详细的结果可以在奥哈拉等地找到。3和 Nachbar等。4。
将在这里使用的 ACE 方法是基于我们以前发布的作品6。然而, 在蛋白质样品中加入 EOF 标记乙酰苯胺是不合适的。AtHIRD11 显示了广泛的峰值模式, 并将 EOF 标记添加到样本将伪装成两个峰值。因此, 标记在单独的运行中使用。在对约束行为进行检验之前, 证实了先前实验中发现的峰值来自不同的构象。因此, CGE 被用来区分蛋白质构象, 转化后的修饰蛋白, 和杂质, 如 AtHIRD11 的片段, 由其不同的质量。然后, 对不同金属离子的特征 AtHIRD11 样品的结合行为进行了研究。
本文的目的是描述一个实验设置, 以区分一个国内流离失所者和其他组成部分的样本, 以评估不同的绑定行为的构象。

<table><tbody><tr><td>AtHIRD11 sample</td><td>Shizuoka University (Group Prof. M. Hara)</td><td>-</td><td>Dehydrin Protein from &lt;em&gt;Arabidopsis thaliana&lt;/em&gt; expressed in &lt;em&gt;Escherichia coli&lt;/em&gt;</td></tr><tr><td>Barefused silica capillary</td><td>Polymicro Technologies (Phoenix, USA)</td><td>106815-0017</td><td>TSP050375, 50 &amp;mu;m inner diameter, 363 &amp;mu;m outer diameter, polyimide coating</td></tr><tr><td>Agilent 1600A</td><td>Agilent Technologies (Waldbronn, Germany)</td><td>comercially not available anymore</td><td>Capillary electrophoresis instrument; Agilent 7100 CE can be used instead</td></tr><tr><td>Agilent 7100 CE</td><td>Agilent Technologies (Waldbronn, Germany)</td><td>G7100A</td><td>Capillary electrophoresis instrument</td></tr><tr><td>Injekt 2 mL</td><td>B. Braun (Melsungen, Germany)</td><td>4606051V</td><td>Syringe for filtration</td></tr><tr><td>Rotilabo-syringe filters</td><td>Carl Roth GmbH + Co. KG (Karlsruhe, Germany)</td><td>KY62.1</td><td>PVDF membrane filter for solution filtration</td></tr><tr><td>Eppendorf Research plus 10 &amp;mu;L</td><td>Eppendorf (Wesseling-Berzdorf, Germany)</td><td>3121 000.023</td><td>Micro pipette for sample handling</td></tr><tr><td>Eppendorf Research plus 10 &amp;mu;L</td><td>Eppendorf (Wesseling-Berzdorf, Germany)</td><td>3121 000.120</td><td>Micro pipette for handling the ligand solutions</td></tr><tr><td>Bulb pipette 10 mL</td><td>Duran Group GmbH(Mainz, Germany)</td><td>24 338 08</td><td>Preparing theNaOH solution</td></tr><tr><td>Bulb pipette 25 mL</td><td>Duran Group GmbH(Mainz, Germany)</td><td>24 338 14</td><td>Preparing the ligand solution</td></tr><tr><td>Duran glas volumetric flask 25 mL</td><td>Duran Group GmbH(Mainz, Germany)</td><td>24 671 1457</td><td>Preparing the ligand stock solution</td></tr><tr><td>Duran glas volumetric flask 10 mL</td><td>Duran Group GmbH(Mainz, Germany)</td><td>24 671 1054</td><td>Preparing the ligand stock solution</td></tr><tr><td>Proteome Lab SDS MW Gel Buffer</td><td>Beckman Coulter (Brea, USA)</td><td>comercially not available anymore</td><td>Separation during capillary gel electrophoresis / Alternative SDS buffer: CE-SDS run buffer from Bio-Rad Laboratories (M&amp;uuml;nchen, Germany) Catalog Number: 1485032&amp;nbsp;</td></tr><tr><td>Acetanilide</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>397229-5G</td><td>Electroosmotic flow marker</td></tr><tr><td>Manganese(II) chloride</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>13217</td><td>Ligand</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>431788-100G</td><td>Rinsing ingredient</td></tr><tr><td>Bariumchloride</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>202738-5G</td><td>Ligand</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>71729-100G</td><td>Solublizing protein for capillary gel electrophoresis</td></tr><tr><td>Nickel(II) chloride hexahydrate</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>654507-5G</td><td>Ligand</td></tr><tr><td>Selenium(IV) chloride</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>323527-10G</td><td>Ligand</td></tr><tr><td>2-amino-2-hydroxy-methylpropane-1.3-diol (Tris)</td><td>Sigma-Aldrich (Steinheim, Germany)</td><td>252859-100G</td><td>Buffer ingredient</td></tr><tr><td>Zinc(II) chloride</td><td>Merck Millipore ( Darmstadt, Germany)</td><td>1088160250</td><td>Ligand</td></tr><tr><td>Strontium nitrate</td><td>Merck Millipore ( Darmstadt, Germany)</td><td>1078720250</td><td>Ligand</td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck Millipore ( Darmstadt, Germany)</td><td>1371015000</td><td>Ligand</td></tr><tr><td>37% hydrochloric acid</td><td>Merck Millipore ( Darmstadt, Germany)</td><td>1003171000</td><td>Adjusting pH</td></tr><tr><td>Copper(II) chloride dihydrate</td><td>Riedel-de Ha&amp;euml;n (Seelze, Germany)</td><td>31286</td><td>Ligand</td></tr><tr><td>Sonorex Longlife RK 1028 CH 45L</td><td>Allpax (Papenburg, Germany)</td><td>10000084;0</td><td>Ultrasonic bath</td></tr><tr><td>Agilent ChemStation Rev. 8.04.03-SP1</td><td>Agilent Technologies (Waldbronn, Germany)</td><td>G2070-91126</td><td>Software packages to operate the CE instruments, acquisite data and evaluate it</td></tr></tbody></table>

analysis of athird11 intrinsic disorder and binding towards metal ions by capillary gel electrophoresis and affinity capillary electrophoresis

植物强烈依赖于他们的环境。为了适应压力变化 (例如干旱和高盐度), 高等植物进化出属于内在紊乱的蛋白质 (国内流离失所者), 以减少氧化和渗透胁迫。本文采用毛细管凝胶电泳 (CGE) 和移动性移位亲和电泳 (ACE) 相结合的方式来描述拟南芥不同构象的 AtHIRD11 的结合行为。CGE 用于确认 AtHIRD11 的纯度, 并排除片段、翻译后修饰和其他杂质作为复杂峰值模式的原因。在这一部分的实验中, 不同的样品成分是由一个粘性凝胶在毛细管内的不同质量和检测与二极管阵列探测器。然后, 用 ACE 研究了样品对各种金属离子的结合行为。在这种情况下, 配体被添加到缓冲区解决方案中, 并测量迁移时间的偏移量, 以确定是否发生了绑定事件。使用 CGE 和 ACE 的组合来确定国内流离失所者的结合行为的优点之一是, 有可能自动进行凝胶电泳和结合试验。此外, 专家咨询小组显示的检测限制比经典凝胶电泳和 ACE 能够确定的方式, 结合一个配体的快速方式。此外, ACE 也可以应用于其他带电的物种比金属离子。但是, 使用这种方法进行绑定实验, 其确定绑定站点数量的能力受到限制。然而, 专家咨询小组和 ACE 的结合可以适应于描述任何蛋白质样品对许多带电配体的结合行为。

图 1显示了在 CGE 实验中获得的 AtHIRD11 样品的电泳图谱。肽的大小从左向右增加。峰值4有最大的质量和表明完整的蛋白质。较小的峰值2和3代表较小的杂质 (如降解产物)。在没有蛋白质样本的情况下, 第一峰值和基线的不一致也可以复制。因此, 它与 SDS 凝胶本身有关, 不代表任何与样品相关的杂质。
<p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis at JoVE.com

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

该协议结合毛细管电泳对蛋白质样品的特性进行了表征, 并对带电配体进行了快速结合筛选。推荐用于具有弹性结构的蛋白质, 如内在紊乱的蛋白质, 以确定不同构象的结合的差异。

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

analysis-athird11-intrinsic-disorder-binding-towards-metal-ions

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

5.9K 次观看.  TU Braunschweig. 该协议结合毛细管电泳对蛋白质样品的特性进行了表征, 并对带电配体进行了快速结合筛选。推荐用于具有弹性结构的蛋白质, 如内在紊乱的蛋白质, 以确定不同构象的结合的差异。

视频: Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

科研

化学

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

用尺寸排阻ICP-MS标准定量分析Metalloproteomic

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

生物化学

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

革兰氏阴性菌的基本金属摄取: x 射线荧光、放射性同位素和细胞分馏

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

金属浸出在固定金属亲和力色谱中的定量

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

使用离子淌度-质谱对Ni（II）和Zn（II）三元配合物进行热化学研究

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been reported to directly bind to a variety of peptides and play both necessary and pathological roles ranging from mediating structure to electron transfer properties to imparting catalytic function. Quantifying the binding affinity and thermodynamics of these Cu(II)-peptide complexes in vitro provides insight into the thermodynamic driving force of binding, potential competitions between different metal ions for the peptide or between different peptides for Cu(II), and the prevalence of the Cu(II)-peptide complex in vivo. However, quantifying the binding thermodynamics can be challenging due to a myriad of factors, including accounting for all competing equilibria within a titration experiment, especially in cases where there are a lack of discrete spectroscopic handles representing the peptide, the d-block metal ion, and their interactions.
Here, a robust set of experiments is provided for the accurate quantification of Cu(II)-peptide thermodynamics. This article focuses on the use of electronic absorption spectroscopy in the presence and absence of chromophoric ligands to provide the needed spectroscopic handle on Cu(II) and the use of label-free isothermal titration calorimetry. In both experimental techniques, a process is described to account for all competing equilibria. While the focus of this article is on Cu(II), the described set of experiments can apply beyond Cu(II)-peptide interactions, and provide a framework for accurate quantification of other metal-peptide systems under physiologically relevant conditions.

Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores

This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

量化Cu（II）和肽残基在存在和不存在发色团的情况下的结合相互作用

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been ...

基于膦酸盐的 MOF 的合成和发现：一种溶热方法

Discovery and Synthesis Optimization of Isoreticular Al(III) Phosphonate-Based Metal-Organic Framework Compounds Using High-Throughput Methods

High-throughput (HT) methods are an important tool for the fast and efficient screening of synthesis parameters and the discovery of new materials. This manuscript describes the synthesis of metal-organic frameworks (MOFs) from solution using an HT reactor system, resulting in the discovery of various phosphonate-based MOFs of the composition [Al2H12-x(PMP)3]Clx&#8729;6H2O (H4PMP = N,N &#39;-piperazine bis(methylenephosphonic acid)) for x = 4, 6, denoted as Al-CAU-60-xHCl, containing trivalent aluminum ions. This was accomplished under solvothermal reaction conditions by systematically screening the impact of the molar ratio of the linker to the metal and the pH of the reaction mixture on the product formation. The protocol for the HT investigation includes six steps: a) synthesis planning (DOE = design of&#160;experiment) within the HT methodology, b) dosing and working with in-house developed HT reactors, c) solvothermal synthesis, d) synthesis workup using in-house developed filtration blocks, e) characterization by HT powder X-ray diffraction, and f) evaluation of the data. The HT methodology was first used to study the influence of acidity on the product formation, leading to the discovery of Al-CAU-60&#8729;xHCl (x = 4 or 6).

作者聚焦：加速微孔材料化学的发现 

The targeted synthesis of new metal-organic frameworks (MOFs) is difficult, and their discovery depends on the knowledge and creativity of the chemist. High-throughput methods allow complex synthetic parameter fields to be explored quickly and efficiently, accelerating the process of finding crystalline compounds and identifying synthetic and structural trends.

基于高通量方法的等网状Al（III）膦酸盐基金属有机骨架化合物的发现与合成优化

High-throughput (HT) methods are an important tool for the fast and efficient screening of synthesis parameters and the discovery of new materials. This ...