Quantifying Lysosomal Degradation of Extracellular Proteins With a Fluorescent Protein-Based Internalization Assay

Endocytosis is an essential membrane transport mechanism that is indispensable for the maintenance of life. It is responsible for the selective internalization and subsequent degradation or recycling of specific extracellular proteins and nutrients, thereby facilitating cellular nutrient supply, modulation of receptor signaling, and clearance of foreign substances. However, methods for the quantitative analysis of lysosomal degradation of extracellular proteins via endocytosis remain limited. This protocol describes a method for purifying the protein-of-interest (POI)-red fluorescent protein (RFP)-green fluorescent protein (GFP) fusion protein, which is modified with specific mammalian cell glycans or other modifications, from the conditioned medium of mammalian cell cultures. Subsequently, the protocol details a quantitative approach for evaluating its internalization and lysosomal degradation within cells using the RFP-GFP tandem fluorescent reporter. Following the addition of POI-RFP-GFP to the medium, cells can be subjected to cell biological assays, such as flow cytometry, as well as biochemical analyses, such as immunoblotting. This protocol is broadly applicable to studies of the internalization of extracellular proteins. Key features • Purification of secreted GFP-RFP-fused POI from mammalian cell culture supernatant. • Quantification of POI-RFP-GFP internalization through measurement of GFP and RFP signals using flow cytometry. • Confirmation of lysosomal degradation of POI-RFP-GFP by immunoblotting.