Home › Biochemistry › The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Biochemistry JoVE (Open Access) Citable · DOI

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

DOI: 10.3791/50867-v
What you'll learn
  • ✓ Perform biotinylation-based endocytic assays on apical membrane domains
  • ✓ Quantify plasma membrane protein internalization and recycling dynamics
  • ✓ Interpret trafficking data from model epithelial cell systems
Protocol

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Difficulty
advanced
Total time
~4–6 hours per assay (including incubation and detection steps)
Model organism
CFBE41o- cells (cystic fibrosis bronchial epithelial)
Biosafety
BSL-1

Steps

1
Biotinylate apical membrane domain proteins

Label surface-exposed proteins on the apical membrane using biotin reagents under non-permeabilizing conditions. This selectively marks proteins available for endocytic uptake.

▶ 01:45
2
Measure CFTR endocytosis in epithelial cells

Monitor cystic fibrosis transmembrane conductance regulator (CFTR) internalization kinetics in CFBE41o- cells following biotinylation, quantifying protein trafficking to intracellular compartments.

▶ 07:54
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