Protein patterning with a photoactivatable derivative of biotin.
A method is described for the covalent immobilization of macromolecules at defined location on polymer surfaces. A thin film of a photoactivatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin, in salt form) was dried onto polystyrene or nitrocellulose surfaces and then exposed to intense white light through a mask to yield patterns of biotin covalently bound to the polymers. The subsequent addition of avidin resulted in the formation of surfaces to which biotinylated molecules could then be bound through a biotin-avidin-biotin bridge. To develop the pattern of avidin on the surfaces, biotinylated enzymes (alkaline phosphatase and horseradish peroxidase) were specifically immobilized on the surface. These enzymes still retained their catalytic activities and were visualized through the formation of colored or fluorescent products. Various factors such as concentration, irradiation time, and light intensity were shown to determine the amount of active enzyme that could be bound and so by implication the degree of photobiotinylation that had occurred.
Hengsakul
M
M
Department of Biochemistry, Imperial College of Science, Technology & Medicine, South Kensington, London, U.K.
Cass
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D000345:Affinity Labels; D000469:Alkaline Phosphatase / Q000032:analysis*; D001360:Avidin / Q000032:analysis*; D001386:Azides / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D003101:Collodion; D004800:Enzymes, Immobilized / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D006735:Horseradish Peroxidase / Q000032:analysis*; D008027:Light; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D011108:Polymers; D011137:Polystyrenes; D017550:Spectroscopy, Fourier Transform Infrared
D000345:Affinity Labels; D001386:Azides; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011108:Polymers; D011137:Polystyrenes; D001360:Avidin; D001710:Biotin; D003101:Collodion; C045549:photobiotin; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D016650:Fluorescein-5-isothiocyanate