Fabrication of Histidine-Tagged Fusion Protein Arrays for Surface Plasmon Resonance Imaging Studies of Protein−Protein and Protein−DNA Interactions
| Autor: | Robert M. Corn, Greta J. Wegner, Gerard Marriott, Hye Jin Lee |
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| Rok vydání: | 2003 |
| Předmět: | |
| Zdroj: | Analytical Chemistry. 75:4740-4746 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format. |
| Databáze: | OpenAIRE |
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