Person: Koehler, Stephan
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Koehler
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Stephan
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Koehler, Stephan
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Publication Label-free single-cell protein quantification using a drop-based mix-and-read system(Nature Publishing Group, 2015) Abbaspourrad, Alireza; Zhang, Huidan; Tao, Ye; Cui, Naiwen; Asahara, Haruichi; Zhou, Ying; Yue, Dongxian; Koehler, Stephan; Ung, Lloyd W.; Heyman, John; Ren, Yukun; Ziblat, Roy; Chong, Shaorong; Weitz, DavidQuantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry.Publication Geometric constraints during epithelial jamming(Springer Science and Business Media LLC, 2018-04-02) Atia, Lior; Bi, Dapeng; Sharma, Yasha; Mitchel, Jennifer; Gweon, Bomi; Koehler, Stephan; DeCamp, Stephen; Lan, Bo; Kim, Jae Hun; Hirsch, Rebecca; Pegoraro, Adrian; Lee, Kyu Ha; Starr, Jacqueline; Weitz, David; Martin, Adam; Park, Jin-Ah; Butler, James; Fredberg, JeffreyPublication A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders(Nature Publishing Group, 2016) Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David; Chong, ShaorongDrop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.