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。在减小注入量时, 建议降低注入压力, 而不是注入时间, 因为该仪器需要一段时间才能达到程序注射压力。
不同的蛋白质样品可以找到异常宽广的峰。这种模式可以通过增加电压来影响。因此, 峰值变窄, 分离时间减少。然而, 较短的分离时间会导致未分辨峰值, 较高的电压会增加焦耳加热, 并通过电泳色散7、8限制窄峰的影响。因此, 必须确定对每种新的蛋白质检查的正确条件。
结合 CGE 和 ACE 收集有关蛋白质内在无序特性的信息及其结合行为是一种新的方法。用凝胶电泳对样品进行表征是证明所观察到的多峰与杂质无关的必要步骤。与经典凝胶电泳9相比, CGE 的优点是自动化它的可能性, 制备时间缩短, 分辨率更高。利用 ace 进行国内流离失所者结合实验表明, 它的优越性, 以其他化验 (如固定化金属亲和层析在结果), 因为 ace 不仅显示绑定事件。此外, 它分离不同的蛋白质构象, 可以揭示不同构象的结合配体的差异。在绑定实验过程中, 没有分离的方法无法检测到此行为。此外, 分析时间仅为6分钟;因此, 可以在短时间内计算出10的结果。另一方面, 快速分析在估计绑定站点11的数量时有局限性。
这种方法的未来在于有可能以快速的方式筛查蛋白质, 特别是国内流离失所者的潜在结合伙伴。它给出了一个示例中各种构象的不同绑定行为的快速概述。由于 ACE 本身不能区分杂质和蛋白质本身, 因此使用尺寸依赖性分离 (例如, CGE) 来进行样本表征是强制性的。然而, 专家咨询小组对不同规模的物种的数量提出了看法。ACE 作为结合试验的应用已经从金属离子配体扩展到任何带电配位12。因此, 它可以添加与任何特征绑定的交互的附加信息, 因为分离和结合分析在一个方法的组合可以证明任何改变的结合行为的不同构象和翻译后修改, 分别。

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 border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">AtHIRD11 sample</td>
			<td style="width:111px;">Shizuoka University (Group Prof. M. Hara)</td>
			<td style="width:119px;">-</td>
			<td style="width:192px;">Dehydrin Protein from Arabidopsis thaliana expressed in Escherichia coli</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Barefused silica capillary</td>
			<td style="width:111px;">Polymicro Technologies (Phoenix, USA)</td>
			<td style="width:119px;">106815-0017</td>
			<td style="width:192px;">TSP050375, 50 &#956;m inner diameter, 363 &#956;m outer diameter, polyimide coating</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Agilent 1600A</td>
			<td style="width:111px;">Agilent Technologies (Waldbronn, Germany)</td>
			<td style="width:119px;">comercially not available anymore</td>
			<td style="width:192px;">Capillary electrophoresis instrument; Agilent 7100 CE can be used instead</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Agilent 7100 CE</td>
			<td style="width:111px;">Agilent Technologies (Waldbronn, Germany)</td>
			<td style="width:119px;">G7100A</td>
			<td style="width:192px;">Capillary electrophoresis instrument</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Injekt 2 mL</td>
			<td style="width:111px;">B. Braun (Melsungen, Germany)</td>
			<td style="width:119px;">4606051V</td>
			<td style="width:192px;">Syringe for filtration</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Rotilabo-syringe filters</td>
			<td style="width:111px;">Carl Roth GmbH + Co. KG (Karlsruhe, Germany)</td>
			<td style="width:119px;">KY62.1</td>
			<td style="width:192px;">PVDF membrane filter for solution filtration</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eppendorf Research plus 10 &#956;L</td>
			<td style="width:111px;">Eppendorf (Wesseling-Berzdorf, Germany)</td>
			<td style="width:119px;">3121 000.023</td>
			<td style="width:192px;">Micro pipette for sample handling</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eppendorf Research plus 10 &#956;L</td>
			<td style="width:111px;">Eppendorf (Wesseling-Berzdorf, Germany)</td>
			<td style="width:119px;">3121 000.120</td>
			<td style="width:192px;">Micro pipette for handling the ligand solutions</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bulb pipette 10 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 338 08</td>
			<td style="width:192px;">Preparing theNaOH solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bulb pipette 25 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 338 14</td>
			<td style="width:192px;">Preparing the ligand solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Duran glas volumetric flask 25 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 671 1457</td>
			<td style="width:192px;">Preparing the ligand stock solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Duran glas volumetric flask 10 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 671 1054</td>
			<td style="width:192px;">Preparing the ligand stock solution</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Proteome Lab SDS MW Gel Buffer</td>
			<td style="width:111px;">Beckman Coulter (Brea, USA)</td>
			<td style="width:119px;">comercially not available anymore</td>
			<td style="width:192px;">Separation during capillary gel electrophoresis / Alternative SDS buffer: CE-SDS run buffer from Bio-Rad Laboratories (M&#252;nchen, Germany) Catalog Number: 1485032&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Acetanilide</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">397229-5G</td>
			<td style="width:192px;">Electroosmotic flow marker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Manganese(II) chloride</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">13217</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">431788-100G</td>
			<td style="width:192px;">Rinsing ingredient</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bariumchloride</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">202738-5G</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium dodecyl sulfate</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">71729-100G</td>
			<td style="width:192px;">Solublizing protein for capillary gel electrophoresis</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nickel(II) chloride hexahydrate</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">654507-5G</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Selenium(IV) chloride</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">323527-10G</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-amino-2-hydroxy-methylpropane-1.3-diol (Tris)</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">252859-100G</td>
			<td style="width:192px;">Buffer ingredient</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Zinc(II) chloride</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1088160250</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Strontium nitrate</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1078720250</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Calcium chloride dihydrate</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1371015000</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">37% hydrochloric acid</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1003171000</td>
			<td style="width:192px;">Adjusting pH</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Copper(II) chloride dihydrate</td>
			<td style="width:111px;">Riedel-de Ha&#235;n (Seelze, Germany)</td>
			<td style="width:119px;">31286</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sonorex Longlife RK 1028 CH 45L</td>
			<td style="width:111px;">Allpax (Papenburg, Germany)</td>
			<td style="width:119px;">10000084;0</td>
			<td style="width:192px;">Ultrasonic bath</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Agilent ChemStation Rev. 8.04.03-SP1</td>
			<td style="width:111px;">Agilent Technologies (Waldbronn, Germany)</td>
			<td>G2070-91126</td>
			<td style="width:192px;">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。
图 2代表了在 ACE 实验中对乙酰苯胺的电泳图谱。乙酰苯胺溶液只显示1高峰值, 因为它不应该有杂质。峰值最大值的检测时间表示渗流 (tEOF) 的迁移时间, 用于计算相互作用。
图 3是在没有 SDS 和金属离子的 ACE 实验中, AtHIRD11 样品的电泳图谱。它显示, 除了2的杂质, 从专家咨询小组电泳图谱, 至少5峰, 这是与蛋白质本身有关。峰值1和2可以被分配到杂质, 因为他们的迁移时间表明, 它们是小的或高度积极的电荷。这个假设由高峰高度和区域的增量支持。由于 AtHIRD11 峰3和4接近 EOF, 他们几乎没有充电, 并且不可能在每奔跑被分离。他们几乎不显示相互作用和代表构象没有容易接近的残余必要为与金属离子的相互作用。由于不同构象的有效半径不同, AtHIRD11 显示了几种不同的电荷-大小比值。这些构象可以连续合并。随后, 峰值 5-7 比通常的蛋白质峰值宽。大峰给出了相互作用动力学的暗示。由于峰不狭窄, 在不同的构象之间的快速和非常缓慢的转换可以被排除。在这两种情况下, 峰值将是基线分离4。在这种情况下, 前平衡步骤可以改变峰值模式, 并能更好地洞察与慢动力学的相互作用。
图 4显示了在6峰值的各种金属离子存在下测量的迁移时间转移的图形化评估。它由计算值ΔR/Rf表示。这个值表明一个转变是多么强烈 (按它的价值) 和蛋白质-金属离子配合物电荷的整体变化 (由它的标志)。为了区分一个重要的相互作用和巧合转移, α = 0.05 的置信区间也被计算出来。在置信区间不与零线相交的情况下, 相互作用被认为是重要的。如果峰值在 10 electropherograms 中至少有 6, 则考虑到结果。峰值6不存在任何运行与500µM Co2 +。每个 AtHIRD11 峰的相互作用的完整列表发表在 Nachbar等的早期著作中。4。
使用迁移时间比ri (在配体的存在下) 和迁移时间比rf (在配体不存在时) 计算ΔR/Rf :
<img alt="Equation 1" src="/files/ftp_upload/57749/57749eq1.jpg">
ri 和rf 的计算使用峰值顶倍t蛋白的蛋白质样品峰值和相应的峰值最高时间为 eof 标记teof: 
<img alt="Equation 2" src="/files/ftp_upload/57749/57749eq2.jpg">
对于ri, 在配体存在的时间使用和为rf, 时间在缺席。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57749/57749fig1.jpg"> 图 1:电泳图谱 AtHIRD11 样品在凝胶缓冲器中的分离.峰值1和其左侧的峰值可以观察, 即使没有样本注射, 所以它们是手工艺品。由于2和3峰值的质量和峰值面积小于4峰值, 因此可能是杂质, 如降解产物。<a href="/files/ftp_upload/57749/57749fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57749/57749fig2.jpg"> 图 2: 电泳图谱的乙酰苯胺的运行.该图只显示了1的峰值, 没有任何金属离子和 SDS 凝胶缓冲剂的化合物。山顶的时间标志着渗的流动。<a href="/files/ftp_upload/57749/57749fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57749/57749fig3.jpg"> 图 3: 例子为样品分离的电泳图谱在 ACE 实验期间显示一个复杂的峰值模式在没有任何金属离子。至少可以确定7个峰值, 并与蛋白质及其2杂质相关。<a href="/files/ftp_upload/57749/57749fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57749/57749fig4.jpg"> 图 4: 用500µM 浓度为6峰值, 对被调查的金属离子的结合行为进行图解评估.ΔR的值给出了 迁移时间漂移的强度, 因此对金属离子结合构象变化的强度有了提示。ΔR的标志表明,与 未绑定蛋白相比, 复合物是否更积极或更负电荷。误差线指示α = 0.05 的置信区间。随后, 它们显示了可以找到真正的ΔRf值的区域, 确定性为95%。红条表示计算所得的数据不足 (峰值在少于 6 electropherograms)。<a href="/files/ftp_upload/57749/57749fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

使用非变性聚丙烯酰胺凝胶电泳人的MxA多亚基蛋白复合物的表征

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

使用中性毛细管单克隆抗体亚型的毛细管电泳分离

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

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 ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...