Analytical Technique for Label-Free Multi-Protein Detection Based on Western Blot and Surface-Enhanced Raman Scattering

Abstract

We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.

Raman and surface‐enhanced Raman scattering in the study of human rotator cuff tissues after shock wave treatment

“…In 2008, a publication offered a ''protocol for label-free protein detection in proteomic research'' based on SERS. [10] The present contribution represents one more step in that direction.…”

Novel chiroptical analysis of hemoglobin by surface enhanced resonance Raman optical activity spectroscopy

“…A similar difference was observed by Han et al in their SERRS study of myoglobin. 38 They showed that the intense 1640 cm 21 peak in the RRS spectrum of Mb disappeared in SERRS spectrum, but a new peak at 1623 cm 21 appeared. Moreover, in SERRS spectrum of Hb we observed a peak with a maximum at 974 cm 21 (vibration of 5 HC 1vinyl bonds in the vinyl radicals of the heme), which can hardly be seen in the RRS spectrum.…”

Fundamentals and applications of SERS-based bioanalytical sensing

“…Acidified [206], (B) capturing agents based on the frequency shift upon the binding of molecules to the antibody [170], (C) convective assembly method used for the controlled aggregation of proteins and AgNPs to obtain rich and reproducible SERS spectra for the label-free protein detection [207], and (D) an approach combining the separation of proteins using PAGE and transferring the protein spots onto cellulose membrane (western-blotting) and detecting with SERS after staining with colloidal AgNPs [208]. Reproduced with permission from Refs.…”

“…An approach combining the separation of proteins using polyacrylamide gel electrophoresis (PAGE) and transferring the protein spots onto a cellulose membrane (western blotting) and detecting with SERS after staining with colloidal AgNPs was reported [208]. Label-free detection of proteins can be performed with differential separation from their mixtures after a convective assembly process.…”