A Chip-based Coimmunoprecipitation Technique For Analysis Of Protein-protein Interactions

Co-Immunoprecipitation is a traditional technique for analysis of protein-protein interactions by the special interactions between antibody and antigen. It can be used to determine physiological interactions of proteins in cells. The disadvantages of co-Immunoprecipitation to detection protein-protein interactions are complicated operation, large errors and difficultly digital results. In the first part, a chip-based co-immunoprecipitation method combining the advantages of the antibody chips with the traditional co-immunoprecipitation has been developed for rapid detection of protein-protein interaction. Nanoliter volumes of anti-Flag M2 antibody are printed on aldehyde modified slides in an array format, which provides the platform for the analysis of immunoprecipitates from small amount of crude cell lysates containing flag-bait and myc-prey. Protein-protein interaction detected through c-Myc tag on the prey protein. Immunofluorescence assay direct use the anti-c-Myc-Cy3 antibody, and then get the signal by fluorescence scanner.At the second part, we developed a visual co-immunoprecipitation chip-based detection technology. The interaction signals were visualized via silver enhancement detection method using biotin tagged anti-Myc antibody followed by streptavidin-labeled gold nanoparticles without any equipment. During silver enhancement, the gold nanoparticles served as nucleation sites for the deposition of metallic silver and the particles grew in size, giving an intensely dark signal which could be visualized with the naked eyes. As a result, the silver enhancement method could produce much high detection sensitivity. Taken together, our results indicate that the visual chip-based coIP technology (vChip-coIP) provides an effective platform for rapid analysis of PPI. In addition, the silver enhancement detection method enables the results be visualized by naked eyes, avoiding the use of expensive fluorescence scanner. vChip-coIP proves to be a simple, user-friendly, cost-effective and highly efficient platform for the comprehensive study of PPI.In the third part, we successfully integrated tyramide signal amplification into silver enhancement method to develop a high-sensitive colorimetric detection approach. We used the double-antibody sandwich mode to detect the human IgG. The target molecules with the HRP enzyme catalytic tyramide form the free radicals, the free radicals coupled with the tyrosine in the vicinity. So every target molecular groups have been marked by a lot of Tyramide, as to achieve signal amplification effect. Combining colloidal gold-silver staining amplification techniques, we used colloidal gold as marker tags, a large number of silver can be deposited on the surface of colloidal gold. The sandwich assay model was employed, and the experimental parameters were systematically optimized, including the concentrations of capture antibody, detection antibody, streptavidin-horseradish peroxidase, biotin-tyramide and streptavidin-nanogold. Furthermore, the quantitative analysis was performed well. The results show that the sensitivity of colorimetric assay could be significantly improved by two-orders of magnitude with tyramide signal amplification system. As a result, this technology provides a new sensitively potential method for the detection of clinical markers.