Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells

Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells

Blog Article

Summary: invertatop squeeze bottle The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation.Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry, suitable to specifically map the content of autophagosomes and potentially other short-lived endomembrane transport vesicles without the need of subcellular fractionation.By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched in membrane-protected compartments can be readily enriched and identified.For complete details on the use and execution of this protocol, please refer spidertattooz.com to Zellner et al.

(2021).