A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study.
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 microg/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.
Bogdanovic
Jelena
J
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.
Colon
Jimmie
J
Baker
Cheryl
C
Huo
Qun
Q
D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000737:chemistry / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D008027:Light; D053768:Metal Nanoparticles / Q000737:chemistry*; D011485:Protein Binding; D012542:Scattering, Radiation
D006046:Gold; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)