Diazo coupling method for covalent attachment of proteins to solid substrates.
We describe a process for covalently linking proteins to glass microscope slides and microbeads in a manner that optimizes the reactivity of the immobilized proteins and that is suitable for high-throughput microarray and flow cytometry analysis. The method involves the diazo coupling of proteins onto activated self-assembled monolayers formed from p-aminophenyl trimethoxysilane. Proteins immobilized by this method maintained bioactivity and produced enhanced levels of protein-protein interaction, low background fluorescence, and high selectivity. The binding of immobilized proteins to their specific binding partner was analyzed quantitatively and successfully correlated with solution concentrations. Diazotized surfaces bound more efficiently to proteins containing a hexahistidine tag than those without a his-tag. Moreover, significantly higher reactivity of the immobilized his-tagged proteins was observed on diazotized surfaces than on amine-terminated surfaces. Results suggest that his-tagged proteins are immobilized by reaction of the his-tag with the diazotized surface, thus offering the possibility for preferential orientation of covalently bound proteins.
Wu
Yang
Y
Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Buranda
Tione
T
Metzenberg
Robert L
RL
Sklar
Larry A
LA
Lopez
Gabriel P
GP
D000818:Animals; D001391:Azo Compounds / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D008863:Microspheres; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D040081:Protein Array Analysis; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties
D001391:Azo Compounds; D010455:Peptides; D011506:Proteins; D012821:Silanes; D049452:Green Fluorescent Proteins; C071717:trimethoxysilane