A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study

• Published in: Analytical biochemistry 2010-10, Vol.405 (1), p.96-102
• Main Authors: Bogdanovic, Jelena, Colon, Jimmie, Baker, Cheryl, Huo, Qun
• Format: Article
• Language: English
• Subjects: Dynamic light scattering; GAPDH; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - chemistry; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - metabolism; Gold - chemistry; Gold nanoparticles; Immunoassay - methods; Light; Metal Nanoparticles - chemistry; Protein aggregates; Protein Binding; Scattering, Radiation
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.06.008

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 μg/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.