Microfluidic Immunoprecipitation for Post-Translational Modified Protein Purification  

作　　者：XIA Hu Bobby Mathew Tom John Hisham Hegab June Feng 

机构地区：[1]Biomedical Engineering Department, Louisiana Tech University, Ruston, USA [2]Engineering Department, Louisiana Tech University, Ruston, USA

出　　处：《Chinese Journal of Biomedical Engineering(English Edition)》2012年第2期80-86,共7页中国生物医学工程学报（英文版）

基　　金：National Institutes of Health and the National Center for Research Resources; grant number: P20RR01645 and NIH grant # DK44510

摘　　要：In this paper, we report an antibody functionalized microimmunopreci- pitation （IX IP） method used for enrich lowabundant post-translational modified （PT~ proteins. The device is fabricated by inert, nontoxic and disposable polydimethylsiloxane （PDMS） using a silane-based chemical modification protocol, which yield antibody- terminated PDMS surfaces. In this study, the IX IP device is specifically designed for the purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model in vitro oxidized bovine serum albumin （BSA） was first derivitized by dinitrophenylhydrazide （DNPH） and then captured by the anti-DNP immobilized on this Ix lP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment Ix IP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.In this paper, we report an antibody functionalized microimmunopreci- pitation(μIP) method used for enrich lowabundant post-translational modified(PTM) proteins. The device is fabricated by inert, nontoxic and disposable polydimethylsiloxane(PDMS) using a silane-based chemical modification protocol, which yield antibody-terminated PDMS surfaces. In this study, the μIP device is specifically designed for the purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model in vitro oxidized bovine serum albumin(BSA) was first derivitized by dinitrophenylhydrazide(DNPH) and then captured by the anti-DNP immobilized on this μIP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment μIP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

关 键 词：post-translational modification microimmunoprecipitation polydi-methylsiloxane microiluidic device 

分 类 号：Q510.3[生物学—生物化学] Q51