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Simplified protocol for detection of protein-ligand interactions via surface-enhanced resonance Raman scattering and surface-enhanced fluorescence.

Analytical chemistry (2008-07-30)
Xiao X Han, Yasutaka Kitahama, Yuhei Tanaka, Jie Guo, Wei Q Xu, Bing Zhao, Yukihiro Ozaki
ABSTRACT

A simple and effective protocol for detections of protein-protein and protein-small molecule interactions has been developed. After interactions between proteins and their corresponding ligands, we employed colloidal silver staining for producing active substrates for surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). Tetramethylrhodamine isothiocyanate (TRITC) and Atto610 were used for both Raman and fluorescent probes. We detected interactions between human IgG and TRITC-anti-human IgG, and those between avidin and Atto610-biotin by surface-enhanced resonance Raman scattering (SERRS) and SEF. The detection limits of the proposed SERRS-based method are comparable to those of the proposed SEF-based one, 0.9 pg/mL for anti-human IgG and 0.1 pg/mL for biotin. This protocol exploits several advantages of simplicity over other SERS and SEF-based related methods because of the protein staining-based strategy for silver nanoparticle assembling, high sensitivity from SERRS and SEF, and high stability in photostability comparing to fluorescence-based protein detections. Therefore, the proposed method for detection of protein-ligand interactions has great potential in high-sensitivity and high-throughput chip-based protein function determination.

MATERIALS
Product Number
Brand
Product Description

Supelco
Atto 610-NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
Sigma-Aldrich
Atto 610, BioReagent, suitable for fluorescence, ≥85.0% (HPCE/HPLC)
Sigma-Aldrich
Atto 610-Biotin, BioReagent, suitable for fluorescence, ≥85% (HPCE)
Sigma-Aldrich
Atto 610 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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