Name: essential metal uptake in gram-negative bacteria: x-ray fluorescence, radioisotopes, and cell fractionation
Uploaded: 2018-02-01T15:00:00.000+00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation at JoVE.com

还收集了通用/CA@APS 的数据, 该基金全部或部分资金来自国家癌症研究所 (ACB-12002) 和国立普通医学科学研究所 (AGM-12006) 的联邦基金会。这项研究使用了先进的光子源 (APS) 的资源, 美国能源部 (doe) 科学办公室的用户设施, 由阿贡国家实验室在合同 No. 操作的能源部科学办公室DE-AC02-06CH11357使用先进的光子源得到了美国能源部, 科学办公室, 基础能源科学办公室的支持, 合同 No。W-31-109-Eng-38
我们要感谢 UAB 综合癌症中心-质谱/蛋白质组共享设施 (P30CA13148-38), 以帮助他们在质谱分析。
C.D.R. 得到了阿拉巴马大学伯明翰分校多元化、公平和包容性办公室的资助。L.L.R. 在伯明翰的阿拉巴马大学放射科的支持下。能源部, 科学办公室, 同位素计划支持52锰生产和 a.v.f. m. 根据授予 DESC0015773。

1. 细菌起动器文化 (天 1)
<ol><li>在200毫升斜角烧瓶, 接种30毫升的 Luria Bertani 汤 (LB) 与大肠杆菌菌株 BL21-CodonPlus (DE3)-RIPL 细胞含有 pYFE3 质粒16。</li>
	<li>加入30µL 50 毫克/毫升氨苄西林, 以吸管和200µL 针尖吸气。在 225 rpm 在37˚C 一夜之间摇晃。</li>
</ol>2. 补充 M9 最低媒体准备 (2 天)
注意: 这是根据 Amresco 手册改编的。
<ol><li>按下列步骤准备 6 L 液体培养基。在 2 l 斜角烧瓶中, 将10.5 克 M9 最小介质添加到超纯 H2o 1 l ˚C 121 分钟, 然后冷却至室温。</li>
	<li>菌添加以下无菌补充解决方案: 2 毫升/升的1米 MgSO4, 10 毫升/升的20% 瓦特/v 葡萄糖, 0.1 毫升/升1米 CaCl2, 1 毫升/升的50毫克/毫升氨苄西林。在生物安全柜中执行此步骤以保持无菌环境。将媒体加热到37˚C。</li>
</ol>3. 细菌亚文化
<ol><li>增加5毫升/升的隔夜起动文化, 以 M9 最小的媒体通过吸用自动吸管和5毫升尖端。在 225 rpm 的37˚C, 连续摇动亚文化的9小时。 注意: 在此步骤中, YfeABCDE 由其本机Y. 耶尔森菌启动子中的 autoinduction 表达。</li>
	<li>通过离心 4500 x g 恢复细胞30分钟在4˚C。重细胞在50毫升的冰冷磷酸盐缓冲液 (20 毫米 Na2HPO4 pH 7.6, 50 毫米氯化钠) 通过吸管和1毫升尖端的吸气, 并冻结过夜在-80 ˚C。</li>
</ol>4. 细胞分馏 (3 天)
<ol><li>解冻悬浮在4° c 和颗粒细胞在 4000 x g 为20分钟在4˚C。重细胞在50毫升冰冷高盐缓冲液 (200 毫米三盐酸 pH 8.0, 400 毫米氯化钠, 和2毫米 EDTA) 通过抽吸与自动吸管和25毫升尖端。将悬浮在冰上孵育20分钟, 偶尔进行混合搅拌。</li>
	<li>在 4500 x g 的20分钟, 在4˚C 的细胞颗粒。重在50毫升冰冷低盐缓冲液 (10 毫米三盐酸 pH 8.0), 通过吸与自动吸管和25毫升尖端的细胞。将悬浮在冰上孵育20分钟, 偶尔进行混合搅拌。</li>
	<li>颗粒的原生质在 4500 x g 为20分钟在4˚C。恢复包含从的上清。重在磷酸缓冲盐水溶液中的颗粒原生质 (步骤 3.2), 通过吸进自动吸管和25毫升尖端, 和溶解细胞三循环的法国压力细胞按 1500 psi。 注: 法国压力电池印刷机可能是笨拙的操作和使用液压泵驱动细胞裂解。使用液压泵时要小心, 确保活塞与压力机的正确对准, 并保持液压泵的免提。</li>
	<li>颗粒的细胞碎片在 5万 x g 20 分钟在4˚C。恢复含有细胞质的上清液。如有必要, 外层和内膜可以进一步分馏16。</li>
</ol>5. 使用 FPLC 的蛋白质纯化
<ol><li>在分馏后立即使用0.45 µm 膜单元过滤周馏分。使用口锁注射器过滤器, 便于快速过滤。平衡5毫升 Q 阴离子交换柱使用 20 mM 的 pH 7.6, 0.05% 瓦特/v 南3加载缓冲区的流量为5毫升/分钟。</li>
	<li>将周滤液加载到预平衡5毫升 q 阴离子交换柱上, 使用 20 mm 的 ph 值 7.6, 0.05% 瓦特/v NaN3加载缓冲区的流量为2毫升/分钟. 继续洗涤5毫升 q 阴离子交换柱使用 20 mM 三 ph 7。6, 0.05% 瓦特/v NaN3加载缓冲区, 直到 A280 的基线读数达到5毫升/分钟的流量。</li>
	<li>洗的束缚蛋白使用20毫米三 pH 7.6, 0.05% 瓦特/v 南3, 0-1 M 氯化钠由线性梯度超过10列容量在一个流量5毫升/分钟. 组合包含280峰值的分数。YfeA elutes 在200毫米氯化钠和300毫米氯化钠之间。</li>
	<li>将洗浓缩到离心浓缩机, 直至体积达到5毫升。</li>
	<li>平衡 Superdex 200 pg 凝胶过滤柱, 20 毫米双 pH 6.3, 50 毫米氯化钠, 0.05% 瓦特/v 南3凝胶过滤缓冲器, 流量为2.5 毫升/分钟。</li>
	<li>将浓阴离子交换洗到预平衡 Superdex 200 pg 凝胶过滤柱上, 用5.5 步流量2.5 毫升/分钟的凝胶过滤缓冲器进行净化. 组合包含与 ~ 30 对应的280峰值的分数"(kDa) 蛋白。 注意: 下面的引用是演示17协议的示例 FPLC 实验。</li>
</ol>6. 蛋白质结晶
<ol><li>浓缩凝胶过滤洗离心浓缩器, 最终蛋白质浓度为22毫克/毫升。<ol>
<li>通过将280读数除以1.276 毫克/毫升来计算蛋白质浓度。专家系统 ProtParam18预测了该理论的消光系数。 注: a280样本读数为 5.104 AU 对应于4.000 毫克/毫升溶液;5.104 AU ÷1.276 毫克/毫升 = 4.00 毫克/毫升。</li>
	</ol>
</li>
	<li>混合1.5 µL 的蛋白质与1.5 µL 的30% 瓦特/v PEG 4000, 50 毫米氯化钠, 20 毫米双-三 pH 6.3, 0.05% 瓦特/v 南3在坐在下降或悬挂下降设置通过吸吸管和10µL 提示。</li>
	<li>孵育结晶下降在 293 K 为 2-4 星期。</li>
	<li>在液态氮气池中的闪光冷冻晶体, 用于运送到同步加速器。</li>
</ol>7. x 射线荧光 (使用 GM/CA 光束软件)
<ol><li>导航到哈奇选项卡. 使用能量副标题将能量设置为10.0 凯文。</li>
	<li>导航到扫描选项卡. 导航到交互式选项卡. 装载示例。</li>
	<li>选择优化荧光信号。在时间副标题下输入4.00 秒。选择取荧光光谱。使用绘图选项卡查看频谱。</li>
</ol>8. 对金属能量吸收峰值的疯狂扫描 (使用 GM/CA 光束软件)
<ol><li>将样品移出光束。导航到扫描选项卡. 导航到周期性表选项卡. 选择感兴趣元素的 K 边。 注意:能量副标题中的能量值在 eV 中。</li>
	<li>导航到哈奇选项卡. 使用能量副标题在凯文的步骤8.1 中设置能量值。</li>
	<li>将样品移入光束。导航到扫描选项卡. 导航到自动选项卡. 在时间副标题下输入2.00 秒。</li>
	<li>选择开始扫描。使用绘图选项卡查看 MAD 扫描。 注意:峰值副标题中的能量值是 eV 而不是凯文。</li>
	<li>选择用荧光完成。</li>
	<li>将样品移出光束。导航到哈奇选项卡. 使用能量副标题将能量值设置为在步骤8.4 中四舍五入到下一个 ev (即,峰值: 9658.3 ev, 设置能量到 9659 ev)。</li>
	<li>将样品移入光束。收集数据集。对所有感兴趣的金属重复步骤 8.1-8.6。</li>
</ol>9. 放射性金属吸收测定的分析协议
<ol><li>在14毫升培养管, 接种5毫升的 LB 与大肠杆菌菌株 BL21-CodonPlus (DE3)-RIPL 细胞含有 pYFE3 质粒16。增加5µL 50 毫克/毫升氨苄西林通过吸管和10µL 尖端的吸气。摇 225 rpm 在37˚C 7 小时。</li>
	<li>在室温下用台式离心机将细胞在 1.5万 x g 处1分钟。在1毫升的补充 M9 最小介质中, 通过吸管和1毫升尖端的吸气来洗涤细胞。重复一次。</li>
	<li>在一个50毫升锥形管, 亚文化细胞到30毫升补充 M9 最小的媒体, 通过吸气吸管和1毫升尖端。摇 225 rpm 在37˚C 9 小时。</li>
	<li>确定每分钟达到大约10万计数所需的放射量 (cpm)。 注: 这将根据用于探测的放射性同位素和仪器而有所不同。例如, 52Mn 中包含 7.4 kilobecquerel (kBq) (0.2 microcurie (µCi)) 的样本给出了一个自动伽玛探测器 (奈) 上的 55万 cpm 的读数。因此, 获得 10万 cpm 计数所需的辐射量可以用以下等式来确定: (10万 cpm/550,000 cpm) * 7.4 kBq (0.2 µCi) = 1.35 kBq (0.036 µCi)。 警告: 放射性衰变导致电离辐射的释放, 这是危险的。当工作与放射线时, 总是使用适当的屏蔽, 并遵循尽可能低的原则, 合理地实现 (低) 通过增加距离和屏蔽, 同时减少曝光时间。只有经过培训的人员才应在专门指定的实验室处理放射性物质, 这些化验室被许可使用放射材料。</li>
	<li>将步骤9.4 中所需的辐射量乘以亚文化的总体积, 以确定所需的放射性总量。增加这个数量 (优选地在小容量 50-100 µL) 对包含亚文化的管子, 以便有 10万 cpm/毫升和漩涡好三十年代。 注: 对于31毫升的亚文化, 所需的辐射总量是 1.35 kBq (0.036 µCi) x 31 毫升 = 41.3 kBq (1.12 µCi)。</li>
	<li>分1毫升的亚文化 (现在包含放射性示踪剂) 到单独1.5 毫升离心管通过吸管和1毫升尖端, 和地方在 37˚C thermomixer, 与涡流在 1 x g. 包括足够的管子, 以便有三复制为每个所需的测量, 以及三额外的复制使用作为标准 (设置的标准预留, 不执行分馏试验的标准)。 注: 在1、2和4小时的化验将需要12管共计。</li>
	<li>在孵化后, 离心管在 1.5万 x g 为三十年代使用台式离心机 (优选地冷却到4˚C) 和摒弃清。</li>
	<li>在1毫升的冰冷, 高盐缓冲 (步骤 4.1) 中, 通过吸进吸管和1毫升尖端, 重新暂停细胞, 并立即在冰上放置20分钟。</li>
	<li>用台式离心机 (优选冷却到4˚C), 再在 1.5万 x g 的三十年代再将细胞重新颗粒, 并丢弃清。 注: 上清液含有未关联的游离金属。</li>
	<li>在冰冷, 低盐缓冲 (步骤 4.2) 中, 通过吸气吸管和1毫升尖端, 重新悬浮细胞, 并在冰上孵育20分钟。 注意: 这是很重要的是通过吸管轻轻吸气, 这一步。</li>
	<li>将细胞在 1.5万 x g 的三十年代, 并收集每个清到新的1.5 毫升离心管。现在每个时间点应该有六根管子。 注: 上清液含有周分数, 颗粒包含膜和细胞质分数。我们指的是颗粒作为细胞质的分数。</li>
	<li>测量每个分数中的放射性 (每个时间点六管), 以及在步骤9.6 中预留的标准。使用标准中的放射性量来确定最初添加到每个样本中的放射性总量。</li>
	<li>为了确定从中放射性吸收的百分比, 请使用以下等式: (平均 cpm 的周分数/平均 cpm 的标准) x 100。</li>
	<li>为了确定在细胞质中放射性摄取的百分比, 请使用以下等式: (标准的细胞质分数/平均 cpm 的平均 cpm) x 100。</li>
	<li>要确定% 总摄取量, 请使用以下等式: (平均 cpm 的周分数 + 平均 cpm 的相应细胞质分数)/(平均 cpm 的标准)) x 100。</li>
</ol>

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作者声明他们没有利益冲突。

细胞分馏是一个有用的工具, 专门探测的内容的蜂窝室, 并可以作为一个有用的工具, 以提取小分子, 如金属原子, 以及大分子, 如蛋白质。值得注意的是, 细胞分馏不是一个绝对的技术, 可能是错误的容易, 不小心注意到混合/悬浮, 孵化温度, 和孵化时间。不完全混合可导致最小的膜裂解, 从而可忽略的分馏, 室温下的分馏可导致两种膜的快速裂解, 导致周分数与细胞质含量的污染, 并延长潜伏期也会导致过度的溶解, 从而造成分数污染。为实现高效和相对完整的外膜裂解 (同时保持内膜) 的合理折衷方法是在低渗分馏缓冲液中, 在冰上进行20分钟的孵化。另一种考虑是提取的方法, 即通过摇晃或涡流的苛刻提取可能会损害提取物的质量, 如导致蛋白质聚集。建议采用刮刀、倒置或吸管吸液等方法进行混合, 以保持高质量的下游分析材料。从制备细胞分馏的纯化可以发生在可变效率, 如图 1所示。在一个实验中, YfeA 开始作为提取的周内容的 8% (图 1C), 而在另一个实验中, YfeA 开始作为17% 的周内容 (图 1D)。这些区别可能起因于几个原因例如变的表达条件 (增加 EDTA 对生长媒介能增加基因的表示由饥饿机制调控, 例如毛皮章程 Yfe 促进者), 反复使用冷冻永久股票和人为错误。而不是调整孵化时间, 建议扩大的准备。从从分数的纯化建议包括一个线性梯度洗脱从阴离子交换色谱步骤。阶梯梯度洗脱是一种洗脱策略, 在流动相组成 (i. e., 0 毫米氯化钠, 50 毫米氯化钠, 150 毫米氯化钠,等) 中使用离散和突变。线性梯度洗脱是一种洗脱策略, 使用渐进变化的流动相组成 (即, 0 毫米氯化钠, 5 毫米氯化钠, 10 毫米氯化钠,等。阶梯梯度是有用的分离分子有不同的亲和力为固定相, 和线性梯度洗脱是有用的分离分子, 有相似的亲和力为固定相。在纯化蛋白的情况下, 没有人工纯化标记 (为了增强对固定相的亲和性) 从一个富含独特蛋白质种类的细胞室, 建议线性梯度洗脱分离蛋白质的非, 可能有相似的亲和力, 固定相作为蛋白质的兴趣。一般来说, 1-2 毫克的纯化载脂蛋白 YfeA 的产量预计从每公升的文化表达 YfeA 从其内源性的鼠疫杆菌启动子。
x 射线荧光是一种技术, 使调查人员能够快速确定样品中的金属含量, 并告知调查人员关于意外的金属合并, 否则将是未知的。虽然 EDTA 是包括在高渗分馏缓冲, 以消除松散相关或自由金属, YfeA 衍生的从部分可以包含一些全息蛋白。鉴于金属运输是一个动态的过程, 也许全息蛋白质的内容来源于 YfeA, 它还没有将其货物捐赠给 YfeBCDE。值得注意的是, X 射线荧光只测量总的金属含量, 不表明金属是否在晶格中有序, 或特定的与蛋白质结合。要确定金属是否在晶格中有序排列, 以及/或特异地与蛋白质分子结合, 需要进行 X 射线散射数据的采集和处理。然而, x 射线荧光可以快速抽样评估金属污染和/或残余的全息蛋白整合。同步辐射时间是有限的, 并没有总有时间来制定一个战略, 收集 x 射线散射数据的每个样本, 因此 x 射线荧光是一个有价值的技术, 以筛选许多晶体和优先级的样品 x 射线应收集散射数据。此外, x 射线荧光可以从不衍射 x 射线的样品中收集数据。在收集 X 射线荧光数据时, 有时信号会非常微弱, 由此产生的光谱言之无物。为了提高信号的强度, 无论是 x 射线荧光或疯狂扫描, 考虑增加曝光时间和/或减少 x 射线束衰减。当样品被确定为收集 x 射线散射数据时, 建议在样品的不同部分收集 x 射线散射数据, 而不是用于 x 射线荧光和疯狂扫描以减少辐射损伤, 提高数据质量收集, 并尽量减少任何潜在的异常信号的减少。
检测放射性示踪剂需要 nanomolar 数量的材料或更少, 并使用这些示踪物作为分子追踪试剂提供了一种简单和高度敏感的探测细胞过程的方法。上述 radiometal 测定方法可用于测定全金属的摄取量, 分布在细胞间, 以及吸收率。事实上, 每个数据的时间点需要40分钟的分馏;然而, 随着每个时间点的到达, 细胞立即颗粒和孵化在高盐缓冲 (图 5, 步骤 1)。Radiometal 测量介质, 丢弃高盐缓冲器 (图 5, 步骤 1) 和周和细胞质分数后 (图 5, 步骤 3) 表示高盐缓冲孵化成功地删除所有剩余的无关联自由金属, 在到达时间点后, 在最初的离心后徘徊。初始离心去除大部分 (高达 80%) 无关联的游离金属, 和离心后, 孵化在高盐缓冲也删除额外剩余的无关联自由金属。在40分钟的分馏过程中, 任何悬而未决的游离金属都不会对金属运输产生显著影响。40分钟的分馏对细胞内金属运输的影响是谨慎的数据解释的另一个考虑。几乎整个分馏协议发生在冰上, 除了30第二次离心之间的步骤。金属运输时间课程实验表明, 保持电池在4° c 废除的金属传输20,21,22;因此, 由于实验是在冰层上进行的, 40 分钟的分馏不会在细胞质的从中显著地增加金属含量, 而金属含量则有望代表细胞的时间点。最初离心。
测定不同细胞间的相对摄取量可以帮助了解 XFS 的数据, 证实基板的生理性质, 并使研究者能够进一步探测一个机制的分子细节。例如, 增加基因突变的 radiometal 吸收实验提供了一个直接的方法来加强关键功能残留物或分子参与基板运输。radiometal 吸收实验和分析的优点包括: 快速周转, 高吞吐量, 高重现性, 以及比较生长条件和遗传结构的实验的并行化。

在革兰氏阴性菌中, 过渡金属原子的细胞质池通过内膜 ABC (ATP 结合盒) 进口商增加和补充, 从而减少了内膜的金属易位。周簇 A-1 基质结合蛋白 (SBPs) 组成一组的伴侣, 直接绑定金属原子和摆渡他们通过从, 以交付他们的同源 ABC 进口商1。其他的 SBPs 群专门用于运输基材, 例如螯合金属 (簇 A-2)、碳水化合物 (b 和 D-1) 和氨基酸 (b、C、F-2 和 F-4);然而, 不管基质, SBPs 遵循一个进化保守的 c 钳折叠2。本文提出的广义的收缩基底转移机制包括 c 钳经历一系列类似金星蝇3的扭曲。一般来说, 集群 A-1 SBPs 纯化的全息 (金属约束) 的形式, 虽然研究这些 SBPs 是有限的困难, 产生的生产力 (无金属) 形式的蛋白质实验4。迄今为止, 研究 A-1 SBPs 的策略仅限于诱变、透析和乙酸酸 (EDTA) 合剂的部分变性,5,6,7,8,9,10,11. 来自这些实验的结构数据表明, 星团 A-1 SBPs 可能不完全遵循金星蝇机制。目前从文献中缺失的是一种使用生理结合的伙伴,在体内生成 A-1 SBPs 的方法。
我们首次使用细胞分馏来纯化 YfeA, 这是鼠疫的病原体, 通过与其生理同源 YfeBCDE ABC 进口商的相互作用, 将其转化为 A-1 形态, 从而使其成为一种由鼠疫耶尔森氏杆菌组成的群. 在牢房里我们通过能量色散 x 射线光谱学 (EDS) 来展示 YfeA 的形式, 也被称为 x 射线荧光。我们还通过将细胞分馏与高度敏感的放射性金属吸收研究相结合, 来证明 YfeBCDE 的功能, 以区分金属在外膜上的摄取和金属在内膜上的进口。使用放射性示踪剂, 可以检测 pg/L 数量的金属, 大大低于检测的限制, 如电感耦合等离子体质谱 (ICP) 或原子吸收光谱 (AAS) 的技术。这允许对所研究的系统进行最小扰动。此外, 所列出的传统方法通常需要更大的样本量、额外的后处理和更长的轮圈时间, 这些都不是示分析的典型。因此, 使用示提供了一个方便和直接的方法来监测金属在细胞内的分布和摄取。还有待确定的是, 如果用这种方法生产的一个载脂蛋白簇 A-1, 将会从目前用于产生蛋白的做法中得到结构观察。
细胞分馏是分离细胞隔间的一种既定方法, 用于在隔离12中专门探测其内容的隐含目的。细胞分馏通常用于确认分子在细胞周期中的位置, 或测量特定隔间的活动13。例如, 金属运输活动可以通过探测金属基板的蜂窝室来检测。整合细胞分馏可以区分和比较金属的摄取横跨外膜和金属转移横跨内膜。这些过程然后可以反过来被测量随着时间。在蛋白质纯化的情况下, 最常见的方法细胞裂解和可溶性成分分离不溶性成分是机械中断 (即研磨, 混合), 高压均质 (即, 法国压力细胞压, 细胞干扰) 和超声波频率 (i. e., 超声)14。使用超声频率溶解细胞通常是最适合小体积;然而, 超声是已知的产生过量的热量, 可以变性蛋白质。为了避免产生过量的热量, 超声频率通常被应用在连续的短脉冲中, 这可能会耗费时间, 并无意间将 DNA 分子分解成更小的碎片。此外, 在准备和分析实验中, 可能需要多个昂贵的超声探头, 因为每个探头的样本量范围有限, 可以进行处理。在寒冷的环境如寒冷的房间里, 使用高压溶解细胞避免在裂解或操作细胞干扰的过程中在细胞裂解时产生过热的压力。像超声探头一样, 可能需要购买多组法国压力电池冲压组件来处理大量的样品卷。法国压力电池按组件是容易连接, 但笨拙的操作和清洁, 可能是沉重的移动和容易堵塞, 从密集的样品。利用超声频率和高压系统对溶解细胞的好处包括高样品回收率, 能处理多样品与最小的交叉污染, 和可调整的剪切力, 可以适应裂解优化多种样本类型。通过调整超声波频率、爆炸持续时间、每平方英寸活塞压力磅 (psi) 和通过压力机的通过次数, 可以实现优化。对溶解细胞的机械破坏可能是最技术上的琐碎和最便宜的细胞裂解策略。机械中断可能会给样品回收带来挑战, 并且不适合处理多个样品。机械破坏比高压或超声波频率更温和, 但不是高吞吐量, 容易产生低效的裂解。一个重要的实验限制机械中断, 高压均匀化, 和超声频率, 是无法控制的破坏整个蜂窝结构, 这样, 在裂解后, 所有的水细胞组分混合一起.此外, 所有的细胞隔间不会同时中断;因此, 溶解早期的舱室的内容可能会受到持续的破坏性力量的干扰, 比溶解晚的舱室的内容还要长。细胞分馏允许便宜, 技术简单, 高效的细胞内容分离的基础上, 同时避免昂贵的设备和样品接触苛刻的, 破坏性的力量的需要。
在这种情况下, 进口的基质被重新引入到潜在的蛋白, 并在裂解过程中再生全息蛋白, 在下游色谱或其他纯化技术之前。在裂解过程中加入 edta 合剂是一个额外的障碍, 在这种情况下, 由于 edta 将金属从色谱树脂中剥离出来并防止蛋白质结合15, 因此无法将金属亲和性作为第一步进行蛋白质纯化。一个简单和廉价的解决方案是细胞分馏渗透休克, 其中差异离心和普通实验室试剂, 使研究员仔细提取足够的蛋白质功能分析。渗透冲击分馏利用渗透压的两个步骤的变化, 以分离的细胞舱室。首先, 高渗缓冲液会使细胞在水离开每个细胞时产生圆齿, 其次, 低渗缓冲液会使细胞膨胀, 因为水重新进入每个细胞。通过仔细控制细胞膨胀的时间, 外层的膜可以有选择地裂解 (由于来自流入水的渗透压的积聚), 从而使它们的内容排空到缓冲液中。内膜的保存保证了细胞质中的原生质, 并通过离心分离与周的含量。
YfeA 是一种能结合铁、锰和锌原子的 polyspecific 的收缩型,16。结合金属的相对比例可以根据生长过程中的金属补充而改变, 并通过 X 射线荧光检测, 如阿贡国家实验室的高级光子源16。x 射线荧光使研究人员能够快速地描述一个广泛的金属组装, 而不需要大的或衍射质量的晶体, 甚至可以从蛋白质沉淀或蛋白质溶液中收集数据。
x 射线荧光可以迅速揭示意想不到的结果, 如, 在这种情况下, 主要 YfeA 基板是锌, 而不是书面的生理基质铁或锰的16。如果在收集 x 射线散射数据之前使用, x 射线荧光可以在实验设计中告知研究者, 例如, 用于反常 x 射线散射数据收集的金属能量边缘。就像确定金属的相对丰度一样, X 射线荧光也可以确定样品中是否缺少金属, 提供了一种快速的方法, 即从全息蛋白中分离含有载脂蛋白蛋白的晶体, 并估计金属信号强度不需要耗时的数据处理。在识别具有强金属信号的样品时, 用于反常 X 射线散射数据收集的精确波长可以通过多波长反常色散 (MAD) 扫描来确定。
本详细的协议旨在帮助新的实验者成功地进行细胞分馏, 并有效利用同步辐射光束的硬件来分析全金属含量, 并推进金属结合蛋白的研究。

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Polyethylene glycol 4000, EMD Millipore</td><td>Fisher</td><td>M1097270100</td><td></td></tr><tr><td>Ampicillin Sodium Salt (Crystalline Powder)</td><td>Fisher</td><td>BP1760-5</td><td></td></tr><tr><td>AMRESCO M9 Medium Broth</td><td>Fisher</td><td>NC9688886</td><td></td></tr><tr><td>Bis-Tris, Fisher BioReagents</td><td>Fisher</td><td>BP301-100</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (Certified ACS)</td><td>Fisher</td><td>C79-500</td><td></td></tr><tr><td>Dextrose (D-Glucose) Anhydrous (Granular Powder/Certified ACS)</td><td>Fisher</td><td>D16-500</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (Crystalline/Certified ACS)</td><td>Fisher</td><td>S311-100</td><td></td></tr><tr><td>Fisher BioReagents Microbiology Media: LB Broth, Miller</td><td>Fisher</td><td>BP1426-500</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate (Crystalline/Certified ACS)</td><td>Fisher</td><td>M63-500</td><td></td></tr><tr><td>Sodium Azide, White Powder</td><td>Fisher</td><td>BP922I-500</td><td></td></tr><tr><td>Sodium Chloride (Crystalline/Certified ACS)</td><td>Fisher</td><td>S271-500</td><td></td></tr><tr><td>Sodium Phosphate Dibasic Anhydrous (Granular or Powder/Certified ACS)</td><td>Fisher</td><td>S374-500</td><td></td></tr><tr><td>Tris Hydrochloride (Small White Flakes/Molecular Biology)</td><td>Fisher</td><td>BP153-500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cryschem Plate</td><td>Hampton</td><td>HR3-158</td><td></td></tr><tr><td>EMD Millipore Amicon Ultra-15 Centrifugal Filter Units</td><td>Fisher</td><td>UFC903024</td><td></td></tr><tr><td>EMD Millipore Millex Sterile Syringe Filters: Durapore PVFD Membrane</td><td>Fisher</td><td>SLHV013SL</td><td></td></tr><tr><td>GE Healthcare HiLoad 26/600 Superdex 200 pg</td><td>Fisher</td><td>28-9893-36</td><td></td></tr><tr><td>GE Healthcare HiTrap IEX HP Prepacked Columns</td><td>Fisher</td><td>17-1154-01</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agilent Technologies BL21-CODON PLUS RIPL CELLS</td><td>Fisher</td><td>230280</td><td></td></tr></tbody></table>

essential metal uptake in gram-negative bacteria: x-ray fluorescence, radioisotopes, and cell fractionation

我们展示了一个可伸缩的方法分离细菌从从细胞质。该方法用于纯化周蛋白的生物物理特性, 并测定周与细胞质间的基底转移。通过仔细地限制从与细胞质分离的时间, 实验者可以提取出感兴趣的蛋白质, 并对每个舱室单独进行分析, 而不必在隔间之间进行污染。从分馏提取的蛋白质, 然后可以进一步分析三维结构确定或底物结合剖面。另外, 这种方法可以在孵化后与示, 以确定总百分比的摄取量, 以及分布的示踪剂 (和因此金属运输) 在不同的细菌隔间。实验与示可以帮助区分生理基质和 artefactual 基质, 如那些引起的 mismetallation。
x 射线荧光可用于发现样品中金属的存在或缺失, 以及测量金属结合物中可能发生的变化, 作为生长条件、纯化条件和/或结晶条件的产物。x 射线荧光也为每种金属提供了一个相对的丰度量, 可以用来确定最佳的金属能量吸收峰, 用于反常的 x 射线散射数据的采集。Radiometal 摄取可以作为一种方法来验证的生理性质的基片检测 X 射线荧光, 以及支持发现的新的基板。

收集了 SDS-页凝胶和凝胶过滤谱, 评价了制备从分离纯化蛋白的质量。全息 YfeA 的纯化已被描述为16;然而, 没有报道 YfeA 的纯化。该方法所描述的纯化 YfeA 的策略使用的重组野生类型结构, 不包含任何种类的亲和标记, 特别是可用于蛋白质免疫 (即,他的标签), 和单克隆抗体识别 YfeA 未被描述。因此, 采用质谱分析法对 YfeA 的纯化进行了验证。为了净化 YfeA 的分馏, 建议使用线性洗脱梯度阴离子交换色谱 (图 1)。线性洗脱梯度显着改善 YfeA 富集从约8% 的从分数到49% 的阴离子交换产品计算密度 (图 1C)。该组合物通过凝胶过滤提高到 61%, 而蛋白的洗脱量与全息蛋白的洗脱量 (图 2) 相同。质谱分析通过检测92.5% 的 YfeA 氨基酸序列、246 YfeA 多肽谱、24种独特的肽和50独特的多肽谱, 验证了 YfeA 的纯化。另外, 为了对比线性梯度产品与产品从阶梯梯度洗脱, YfeA 是纯化的阴离子交换使用的洗涤步骤50毫米氯化钠然后洗脱步骤250毫米氯化钠 (图 1D)。阶梯梯度略微丰富了 YfeA 从组成17% 的从馏分到18% 的阴离子交换产品计算的密度。阶梯梯度洗脱还丰富了蛋白质污染带, 质谱鉴定为包含多个大肠杆菌 SBPs 的类似分子量. 由于 HiLoad 26/600 Superdex 200 pg 的分辨率限制, 在污染 SBPs 的分子量和结构上的相似性, 凝胶过滤略有改善, YfeA 浓缩到凝胶过滤产品的20%。如果阴离子交换的线性洗脱梯度是无法达到的, 建议在 25-50 毫米氯化钠增量中探索多步冲洗, 以确定去除这些污染物所需的氯化钠浓度。
纯化的载脂蛋白和全息 YfeA 在相同的结晶条件下结晶, 尽管载脂蛋白和全息 YfeA 晶体形态的图像显示了在亚洲生产力和全息 YfeA 状态之间晶体生长的差异 (图 3)。除质谱验证外, 通过该方法纯化的蛋白质晶体中的 x 射线衍射数据 (未显示) 证实了 YfeA 的提纯和结晶。通过捕获 X 射线荧光光谱 (图 4) 可以评估隔间污染程度 (细胞质锌还原为从分数中的全息蛋白)。鉴于锌是与重组 YfeA16相结合的主要基质, x 射线荧光光谱可以迅速区分含有与全息 YfeA (图 4A) 相称的强锌信号的 YfeA 样品, 表明交叉污染, 或微弱的锌信号暗示的载脂蛋白 YfeA 和最小的交叉污染 (图 4B)。M9 最小介质的电感耦合等离子体质谱分析确认过渡金属含量在亚 micromolar 范围内 (未显示);因此, 在成功的分馏之后, 应该能检测到少量的过渡金属。分析分馏 (图 5) 可用于测量 radiometal 传输速率和捕获功能数据。一个60分钟的快照比较周和细胞质分数的大肠杆菌细胞表达 YfeA 只, 完整的 YfeABCDE 转运器和没有 YfeABCDE 成分揭示的后果, 表达一个单一的重组蛋白或金属摄取的蛋白质复合物 (图 6)。没有表达重组蛋白的大肠杆菌细胞, 将从中的大约一半的52锰添加到介质中, 并将其保留在最小的细胞质中。这种负控制代表内生锰运输。表达重组 YfeA 的大肠杆菌细胞, 将大约四分之一的52锰在培养基中添加到细胞质中, 再在从中保留四分之一。在无 Yfe 转运体的情况下, 从中的锰滞留和有限的运输进入细胞质表明了生产 YfeA 的代谢需求和瓶颈。表达重组 YfeABCDE 转运体的大肠杆菌细胞将近100% 的52锰添加到介质中, 并在从中最小保留。将锰转运到细胞质中, 说明了 Yfe 转运体在锰运输中的作用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57169/57169fig1.jpg"> 图1。YfeA 的分离纯化.A. Yfe 传送器的假设模型。B. 利用阴离子交换的线性梯度洗脱法从从馏分中提取 YfeA 纯化的 SDS 页凝胶。SDS-页凝胶显示丰富的 YfeA (30 kDa) 跨纯化步骤。黑色箭头表示 YfeA 的电泳迁移位置。右侧显示的分子量标准。(1) 从分数。(2) Q 阴离子交换柱流过。(3) Q 阴离子交换柱峰洗脱。(4) Superdex 200 pg 凝胶过滤柱峰值。C. YfeA 浓缩--很好的例子。从B中的 SDS-页凝胶的图像处理计算 YfeA 的总富集量, 从周馏分的8% 增加到凝胶过滤产品的61%。蓝色方框表示用于密度计算的 YfeA/总信号。在车道4中检测到 259/280 YfeA 氨基酸的质谱分析, 在绿色亮点中呈现。氨基酸存在于成熟的多肽中以黑体大写的黑文本表示, 而氨基酸组成的劈开信号肽用斜体小写的灰色文本表示。D. YfeA 浓缩-坏例子。利用阴离子交换的阶梯梯度洗脱, 从从馏分中提取 YfeA 纯化的 SDS 页凝胶。SDS-页凝胶显示丰富的 YfeA 跨纯化步骤。黑色箭头表示在阴离子交换色谱中增强的主要蛋白质污染物的电泳迁移位置。右侧显示的分子量标准。(1) Superdex 200 pg 凝胶过滤柱峰值。(2) Q 阴离子交换柱250毫米氯化钠台阶梯度洗脱。(3) 从分数。SDS-页凝胶的图像处理从D计算 YfeA 的边际总富集, 其密度为最大20% 的凝胶过滤产品。蓝色方框表示用于密度计算的 YfeA/总信号。质谱分析的带黑箭头在车道1检测到26独特的肽 LivJ (39 kDa), 25 独特的肽 EfeO (41 kDa), 和17独特的肽 LivK (39 kDa)。所有三种污染物均周大肠杆菌SBPs.<a href="//cloudflare.jove.com/files/ftp_upload/57169/57169fig1large.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/57169/57169fig2.jpg"> 图2。YfeA 和全息 YfeA 凝胶过滤谱.黑色箭头表示空卷的位置。YfeA (黑色曲线) 和全息 YfeA (蓝色曲线) 的凝胶过滤峰显示两种蛋白状态洗在同一洗脱量, 表明从从馏分纯化的载脂蛋白 YfeA 具有类似的水动力半径, 因为全息 YfeA 从总在法国新闻之后的细胞含量。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57169/57169fig3.jpg"> 图3。YfeA 和全息 YfeA 晶体形态.全息 YfeA 结晶为一个整体晶体, 而载脂蛋白 YfeA 一般在相同的结晶条件下产生姊妹晶体。<a href="//cloudflare.jove.com/files/ftp_upload/57169/57169fig3large.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/57169/57169fig4.jpg"> 图4。YfeA 和全息 YfeA x 射线荧光光谱.A. 全息 YfeA 的 X 射线荧光光谱已从 M9 最小介质中纯化, 没有额外的过渡金属补充, 绿色曲线。特征峰标记为锰、铁和锌。B. 在 YfeBCDE 的背景下产生的 YfeA 样品的 X 射线荧光光谱。特征峰标记为锰、铁和锌。蓝色曲线。YfeA 的 X 射线荧光光谱从 YfeBCDE 的角度纯化, 并成功地分离出从分数, 并对细胞质过渡金属和/或全息 YfeA 蛋白的污染极小。红色曲线。YfeA YfeBCDE 的 X 射线荧光光谱, 从过度溶解和细胞质过渡金属和/或全息 YfeA 蛋白的严重污染中分离出不成功的分馏。<a href="//cloudflare.jove.com/files/ftp_upload/57169/57169fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57169/57169fig5.jpg"> 图5。用于分馏的广义工作流.<a href="//cloudflare.jove.com/files/ftp_upload/57169/57169fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57169/57169fig6.jpg"> 图 6. 60 分钟放射性金属吸收分馏数据.每个实验进行三次, 误差条表示标准偏差。红色栏。大肠杆菌不含 Yfe 转运体的细胞, 在60分钟后将大约50% 的52锰迁移到细胞质中, 并保持低水平的周52. 大肠杆菌细胞, 包含 Yfe 转运体运输 > 90% 的52锰进入细胞质60分钟后, 保持低水平的周52绿条。大肠杆菌只包含 YfeA 传输约25% 的52mn 到细胞质中的单元格, 并在60分钟后的从中保留52mn 的25%。
在这项工作中使用的 radiometal, 52Mn (t1/2 = 5.59 d), 是在 UAB 回旋加速器设施 (伯明翰, AL) 的质子轰击自然 Cr 目标, 并纯化如先前报告的19。警告: 52Mn 是正电子发射器 (β+avg = 242 凯文, 29.4%), 并且还发射了数高能量伽马射线与高强度 (Eγ = 744.2, 935.5, 1434.0 凯文;I = 90.0%, 94.5%, 100%)。因此, 必须按照上述低的辐射防护原则进行实验。所有被指定为放射性废料的物品都应适当地加以控制, 并按照既定程序加以标示和丢弃。

Watch this Scientific Journal Video about Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation at JoVE.com

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

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

本文提出了一种周过渡金属伴侣在其本机结合的背景下提取的协议, 并给出了 X 射线荧光和 radiometal 吸收对其基质含量的生物物理表征。

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

essential-metal-uptake-gram-negative-bacteria-x-ray-fluorescence

Research

JoVE Journal

Chemistry

9.1K 次观看.  University of Alabama at Birmingham. 本文提出了一种周过渡金属伴侣在其本机结合的背景下提取的协议, 并给出了 X 射线荧光和 radiometal 吸收对其基质含量的生物物理表征。

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

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

科研

环境科学

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

化学

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

生物化学

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

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 ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

免疫与感染

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

内塞里亚淋病的金属有限生长，用于金属反应基因特征和从宿主配体中获取金属

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

X-ray Fluorescence (XRF)

Source: Laboratory of Dr. Lydia Finney &#8212; Argonne National Laboratory
X-ray fluorescence is an induced, emitted radiation that can be used to generate spectroscopic information. X-ray fluorescence microscopy is a non-destructive imaging technique that uses the induced fluorescence emission of metals to identify and quantify their spatial distribution.