High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins
High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins
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DOI：
10.1007/978-1-0716-0163-1_5
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发表时间：
2020-01-01
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影响因子：
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通讯作者：
Schaeffer, Patrick M.
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文献类型：
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作者：
Sorenson, Alanna E.;Schaeffer, Patrick M.
差示扫描荧光法可用于各种各样的应用，包括蛋白质功能的表征，结构-活性关系，药物筛选，以及蛋白质纯化，酶活性和结晶的缓冲条件优化。经典差示扫描荧光法的一个局限性是它依赖于高度纯化的蛋白质样品。通过gfp标记蛋白（DSF-GTP）的差示扫描荧光法克服了这一限制。DSF-GTP通过其对GFP荧光的近端扰动效应，专门测量与GFP融合的靶蛋白的展开和聚集。由于这种独特的原理，DSF-GTP可以在其他蛋白质存在的情况下特异性地测量目标蛋白质的热稳定性。此外，GFP提供了一种独特的分析质量控制措施。在这里，我们描述了在96孔板格式下执行DSF-GTP实验的工作流程、步骤和重要注意事项。
Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.