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首页> 外文期刊>Combinatorial Chemistry & High Throughput Screening >Peptide Protein Binding Assay Using ImageFlashPlates or Imaging Beads
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Peptide Protein Binding Assay Using ImageFlashPlates or Imaging Beads

机译：使用ImageFlashPlates或成像珠子进行肽蛋白结合测定

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摘要

Imaging devices used for the measurement of radioligand-receptor binding assays are typically based on charge-coupled device (CCD) cameras, which are more sensitive for red-shifted scintillation. In the past, red-shifted scintillants had only been integrated into microspheres, referred to as scintillation proximity assay (SPA) Imaging Beads+. More recently, ImageFlashPlates+ have been developed that emit light at 615 nm when exposed to β-radiation. In this article, we report the establishment of peptide-protein binding assays using either streptavidin-coated ImageFlashPlates or Imaging Beads in a low volume 384-well format. In these assays, we employed a biotinylated peptide X and a [33P]-phosphorylated protein Y as the binding partner. The FlashPlates required a washing step, the bead-filled microtiter plates (MTPs) needed a centrifugation step for optimal performance in the scintillation measurements. Both the peptide X-loaded FlashPlates and the beads displayed saturable binding of [33P]-phosphorylated protein Y with a similar scintillation efficiency. A KD value of about 30 nmol / l was measured using the bead-based assay. Due to the washing step in the FlashPlate experiment, approximately two-thirds of the [33P]-phosphorylated protein Y were withdrawn from equilibrium binding. This resulted in correspondingly lower scintillation signals for the FlashPlate experiment. For this reason, the FlashPlate produced a Z' value of 0.64 that was lower than the Z' value of 0.87 for the beads. Using a reference inhibitor in a competition assay produced similar IC50 values for the bead-based assay as for the FlashPlate. Depending on the local automation environment either the centrifugation step for the beads or the washing step for the FlashPlates may be considered more or less of a challenge. Low volume 384-well highthroughput screening (HTS) applicable assay formats are achievable using either the ImageFlashPlates or the Imaging Beads.
机译：用于测量放射性配体-受体结合测定的成像设备通常基于电荷耦合设备（CCD）相机，该相机对红移闪烁更为敏感。过去，红移闪烁剂仅整合到微球中，称为闪烁邻近测定（SPA）Imaging Beads +。最近，已经开发出ImageFlashPlates +，当暴露于β射线时会发出615 nm的光。在本文中，我们报告了使用链霉亲和素包被的ImageFlashPlates或成像珠的小体积384孔格式建立了肽-蛋白质结合测定法。在这些测定中，我们采用生物素化的肽X和[33P]磷酸化的蛋白Y作为结合伴侣。 FlashPlates需要清洗步骤，填充珠子的微量滴定板（MTP）需要离心步骤以实现闪烁测量的最佳性能。载有肽X的FlashPlates和珠子均显示出[33P]-磷酸化蛋白Y的饱和结合，并具有相似的闪烁效率。使用基于珠的测定法测得的KD值约为30 nmol / l。由于FlashPlate实验中的洗涤步骤，大约三分之二的[33P]-磷酸化蛋白Y从平衡结合中退出。这导致FlashPlate实验的闪烁信号相应降低。由于这个原因，FlashPlate产生的Z'值为0.64，低于小珠的Z'值为0.87。在竞争分析中使用参考抑制剂可产生与FlashPlate相似的基于珠的分析IC50值。根据当地的自动化环境，或多或少地会考虑珠子的离心步骤或FlashPlates的洗涤步骤。使用ImageFlashPlates或Imaging Beads可实现小体积384孔高通量筛选（HTS）适用的测定形式。

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