Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions.
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.
Wegner
Greta J
GJ
Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706, USA.
Lee
Hye Jin
HJ
Marriott
Gerard
G
Corn
Robert M
RM
D001483:Base Sequence; D006639:Histidine / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D011506:Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine