A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study
A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study
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DOI：
10.1016/j.ab.2010.06.008
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发表时间：
2010-10-01
影响因子：
2.9
通讯作者：
Huo, Qun
中科院分区：
文献类型：
--
作者：
Bogdanovic, Jelena;Colon, Jimmie;Huo, Qun
蛋白质复合体/聚集体及其形成过程的检测、分析和理解对于生物分子研究、诊断和生物制药开发具有极其重要的意义。遗憾的是，可以方便地用于蛋白质复合体/聚集体检测和分析的技术非常有限。将金纳米粒子免疫探针与动态光散射(DLS)相结合，建立了一种用于蛋白质聚集体检测和研究的无标记纳米粒子聚集免疫分析(NanoDLSay)。3-磷酸甘油醛脱氢酶(GAPDH)是蛋白质印迹中常规使用的一种蛋白质靶标，作为负载控制，这里以此为例进行了演示。通过这项研究，我们发现GAPDH在浓度为10-25微克/毫升的特定缓冲溶液中有很强的形成大聚集体的倾向，金纳米粒子免疫探针的强光散射性能大大提高了动态光散射检测蛋白质络合物/聚集体的灵敏度。与用于研究蛋白质络合物和聚集的荧光技术相比，在NanoDLSay中不需要用荧光探针分子标记蛋白质靶标。NanoDLSay是一种非常方便和灵敏的蛋白质复合体/聚集体检测和研究工具。(C)2010 Elsevier Inc.保留所有权利。
The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 mu g/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study. (C) 2010 Elsevier Inc. All rights reserved.