title: A Protocol for Protein Profiling Using Chemoselective Cleavable Linker Probes in Semi-permeabilized Cells.
abstract: Activity-based protein profiling using activity-based probes (ABPs) resulted in the identification of various enzymes that are involved in the onset and progress of diseases. Detection of such proteins, often expressed at low abundance, is greatly enhanced by incorporating chemically cleavable linkers in the ABP of choice. Initial affinity purification, followed by tailored chemical cleavage of the linker, allows for specific release of the captured enzymes and their interaction partners. When the ABPs are delivered directly to semi-permeabilized cells, in contrast to a crude cell lysate, the sensitivity and efficacy of cell impermeable probes can be enhanced even further.
authors: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK.
authors: Jasper H L
authors: JH
authors: Claessen
authors: Chemical Biology, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747AG, Groningen, The Netherlands. m.d.witte@rug.nl.
authors: Martin D
authors: MD
authors: Witte
mesh_terms: D002463:Cell Membrane Permeability; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D015335:Molecular Probes; D011506:Proteins
keywords: Activity-based probes; Chemical cleavage; Cleavable linkers; Delivery; Perfringolysin O; Semi-permeable cells