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Updated: Aug 20, 2026
Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions
Published on: January 5, 2024
Stable transfection of MDCK cells with epitope-tagged human PepT1
Chun Chu1, Curtis T Okamoto, Sarah F Hamm-Alvarez
1Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089-9121, USA.
Purpose:
Establish and characterize a MDCK cell line that stably expressed PepT1.
Methods:
MDCK cells stably transfected with N-terminal HA-tagged hPepT1 were examined by immunostaining using antibodies to HA and hPepT1. HA-hPepT1 expression was verified by immunoprecipitation and Western blotting from cell lysates. The function of the expressed HA-hPepT1 was evaluated by glycyl-sarcosine uptake. The half-life of PepT1 on the cell surface was measured by biotinylation and chase-labeling of the cells, followed by immunoprecipitation with the anti-PepT1 antibody and blotting with HRP-streptavidin.
Results:
Immunolabeling with antibodies to HA and PepT1 showed fluorescence colocalization. Immunoprecipitation with the anti-HA antibody and Western blotting with the anti-PepT1 antibody showed a broad 90-105 kDa band, and vice versa when the antibodies were utilized in the reverse order. Glycyl-sarcosine uptake was increased in the stably transfected cells by 2-5 fold over untransfected control cells. The Km value of 375 microM accurately reflected the characteristic low affinity of PepT1. Finally, the half-life of the surface biotinylated HA-hPepT1 was measured to be 22 hrs.
Conclusions:
A MDCK cell line stably expressed intact and functional HA-tagged hPepT1 on its cell surface. This cell line represents an alternative to the use of Caco-2 cells to evaluate PepT1 function.
