Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip.
A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated.
Chung
Woo-Jae
WJ
School of Chemical Engineering, Seoul National University, Kwanak-Gu, Seoul, Korea.
Kim
Min-Soo
MS
Cho
Suhyung
S
Park
Sung-Soo
SS
Kim
Jong-Ho
JH
Kim
Yong-Kweon
YK
Kim
Byung-Gee
BG
Lee
Yoon-Sik
YS
D001710:Biotin; D002846:Chromatography, Affinity / Q000379:methods*; D056656:Lab-On-A-Chip Devices; D046208:Microchip Analytical Procedures / Q000379:methods*; D008863:Microspheres; D008904:Miniaturization; D010782:Photolysis; D011506:Proteins / Q000302:isolation & purification*; D012313:RNA; D019809:Streptavidin / Q000302:isolation & purification
D011506:Proteins; D012313:RNA; D001710:Biotin; D019809:Streptavidin