Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.
Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.
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DOI：
10.1021/acs.analchem.6b02528
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发表时间：
2017-01-17
影响因子：
7.4
通讯作者：
Hollfelder F
中科院分区：
文献类型：
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作者：
Gielen F;Butz M;Rees EJ;Erdelyi M;Moschetti T;Hyvönen M;Edel JB;Kaminski CF;Hollfelder F
对划分为单个纳升液滴的试剂进行荧光各向异性测量可产生高分辨率结合曲线，从中可以推断出蛋白质-肽相互作用的精确解离常数 (Kd)。使用当前平台，每分钟可进行四次滴定（每次基于约 100 个数据点），化学计量跨度超过 2 个数量级，并且仅需要数十微升试剂。除了使用纯化组分进行亲和力测量外，无需事先了解表达蛋白的浓度即可获得粗制细胞裂解液中未纯化蛋白的 Kd 值，从而避免蛋白纯化。最后，我们展示了如何设置竞争测定来执行重点库筛选，以便不再需要化合物标记。这些数据证明了液滴室在生物分子相互作用的定量表征中的实用性，并建立了荧光各向异性成像作为小型化液滴格式的定量技术，该技术被证明与宏观试管等效物一样可靠。
Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein–peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.