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Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody‐Based Recycling Assays

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin‐dependent endocytosis, represented by the transferrin receptor, and by clathrin‐independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin‐dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC‐conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti‐Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody‐capture protocols. J. Cell. Physiol. 232: 463–476, 2017. © 2016 Wiley Periodicals, Inc.