title: In-gel expression and in situ immobilization of proteins for generation of three dimensional protein arrays in a hydrogel matrix.
abstract: A method has been developed for the direct conversion of DNA arrays into three dimensional protein arrays on a hydrogel matrix. An agarose gel embedded with bacterial protein synthesis machinery was used as the DNA-programmable expression gel matrix for the in situ translation of genes on a DNA array. Upon incubation of the expression gel matrix cast on a DNA array, protein synthesis took place at the interface of the two surfaces and the cell-free synthesized proteins were deposited on the gel matrix surrounding the corresponding DNA spots. Diffusional dilution of the expressed proteins was minimized by modifying the agarose with Ni-NTA moieties. This procedure resulted in the generation of localized protein spots with confined radii. The developed approach not only simplifies the procedures typically used for the preparation of protein arrays but it also provides conditions for the loading of higher amounts of proteins on the array while retaining their structural integrity and functionality over extended time periods.
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Ju-Young
authors.initials: JY
authors.lastname: Byun
authors.forename: Kyung-Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Ka-Young
authors.initials: KY
authors.lastname: Lee
authors.forename: Min-Gon
authors.initials: MG
authors.lastname: Kim
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D000588:Amines / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007118:Immunoassay; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry
chemical_list: D000588:Amines; D055767:Immobilized Proteins; D011506:Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D004247:DNA; D012685:Sepharose; D009571:Nitrilotriacetic Acid