Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits
Tobias Baumann,
Oliver Vosyka,
Bogdan Florea,
Hermen Overkleeft,
Silke Meiners,
Ilona Kammerl
Affiliations
Tobias Baumann
Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Oliver Vosyka
Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bogdan Florea
Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Hermen Overkleeft
Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Silke Meiners
Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Ilona Kammerl
Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ABP Biotin-Epoxomicin (Bio-EP). Covalent labeling of the active catalytic subunits with Bio-EP is followed by a pull-down using streptavidin-coated beads. After elution from the beads, enriched subunits may be detected via Western blot, tandem mass spectrometry (Li et al., 2013), or alternative techniques.
