Articles by Sharon Waichman in JoVE
Other articles by Sharon Waichman on PubMed
기능 Immobilization와 전송 효소 반응에 의해 단백질의 패턴화 합니다 Analytical Chemistry. Feb, 2010 | Pubmed ID: 20092261 기능성 immobilization와 마이크로-및 nanopatterns 단백질의 측면 조직 bioanalytical 및 바이오 소자 miniaturizing에 대 한 중요 한 전제 조건입니다. 효율적인 사이트 관련 단백질 immobilization 기반 효소 phosphopantetheinyl 전송 (PPT)에서 코엔자임 (CoA) 접근, 여기 보고-특정 펩 티 드 태그에 유리 형 표면 functionalized. 우리 분자 폴 리 (에틸렌 글리콜) 폴리머 브러시에 Coa를 밀도가 커플링에 대 한 상향식 표면 개 질 방법을 고안 했습니다. PPTase Sfp에 대 한 다른 대상 펩 티 드를 융합 단백질의 사이트별 효소 immobilization 라벨 무료 실시간 감지에 의해 확인 됐다. 정량적 단백질 단백질 상호 작용 실험 크게 움직이지 단백질의 50%는 완벽 하 게 활성을 확인 했다. 메서드는 성공적으로 다른 단백질을 함께 적용 됩니다. 그러나, immobilization PPT 기반의 다른 immobilization 효율성에는 N-와 C-테르미니 단백질의 융합 되는 다른 펩 티 드 태그에 대 한 관찰 되지 않았다. 이 동원 정지 방법에 근거 하 여 우리 기능 바이너리 마이크로 구조에 단백질의 photolithographic 패턴화 설립.
Maleimide Photolithography for Single-molecule Protein-protein Interaction Analysis in Micropatterns Analytical Chemistry. Jan, 2011 | Pubmed ID: 21186833 Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.
Reconstitution of Membrane Proteins into Polymer-supported Membranes for Probing Diffusion and Interactions by Single Molecule Techniques Analytical Chemistry. Sep, 2011 | Pubmed ID: 21838222 We have established a robust and versatile analytical platform for probing membrane protein function in a defined lipid environment on solid supports. This approach is based on vesicle capturing onto an ultrathin poly(ethylene glycol) (PEG) polymer brush functionalized with fatty acid moieties and subsequent vesicle fusion into a contiguous membrane. In order to ensure efficient formation of these tethered polymer-supported membranes (PSM), very small unilamellar vesicles (VSUV) containing fluorescent lipids or model transmembrane proteins were generated by detergent depletion with cyclodextrin. Thus, very rapid reconstitution of membrane proteins into PSM was possible in a format compatible with microfluidics. Moreover, surfaces could be regenerated with detergent solution and reused multiple times. Lipid and protein diffusion in these membranes was investigated by fluorescence recovery after photobleaching, single molecule tracking, and fluorescence correlation spectroscopy. Full mobility of lipids and a high degree of protein mobility as well as homogeneous diffusion of both were observed. Quantitative ligand binding studies by solid phase detection techniques confirmed functional integrity of a transmembrane receptor reconstituted into these PSM. Colocomotion of individual ligand-receptor complexes was detected, demonstrating the applicability for single molecule fluorescence techniques.
Diffusion and Interaction Dynamics of Individual Membrane Protein Complexes Confined in Micropatterned Polymer-supported Membranes Small (Weinheim an Der Bergstrasse, Germany). Feb, 2013 | Pubmed ID: 23109503 Micropatterned polymer-supported membranes (PSM) are established as a tool for confining the diffusion of transmembrane proteins for single molecule studies. To this end, a photochemical surface modification with hydrophobic tethers on a PEG polymer brush is implemented for capturing of lipid vesicles and subsequent fusion. Formation of contiguous membranes within micropatterns is confirmed by scanning force microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolved single-molecule tracking and localization microscopy. Free diffusion of transmembrane proteins reconstituted into micropatterned PSM is demonstrated by FRAP and by single-molecule tracking. By exploiting the confinement of diffusion within micropatterned PSM, the diffusion and interaction dynamics of individual transmembrane receptors are quantitatively resolved.