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Updated: Feb 26, 2026
Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation
Published on: July 3, 2017
Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of
Vineet Kumar1, Tot Bui Nguyen1, Beáta Tóth2
1Department of Pharmaceutics, University of Washington, P. O. Box 357610, Seattle, Washington, 98195, USA.
Abstract:
Quantitative proteomics, using LC-MS/MS, is increasingly used to quantify drug transporters present in tissues and cells. Most of these investigations quantify total transporter expression in the cells by utilizing a total membrane fraction, not only the plasma membrane. Here, we report development and optimization of a biotinylation method to quantify protein expression of transporters in the plasma membrane of cells. The Pierce cell surface isolation protocol was optimized for plasma membrane isolation. Incubation of OATP1B1-expressing CHO cells with 0.78 mg/mL of membrane impermeable biotinylation reagent (sulfo-NHS-SS-biotin) at 37°C for 1 h resulted in optimum isolation of the plasma membrane. Subsequently, the expression of transporters in the plasma membrane as a percent of the total was determined by quantitative proteomics using LC-MS/MS. Mean (±SD) plasma membrane expression of OATP1B1 in plated OATP1B1-expressing CHO, MDCKII, and HEK293 cells was found to be 79.7% (±4.7%), 67.7% (±12.2%), and 65.3% (±6.8%) of total cell OATP1B1 expression. Mean (±SD) plasma membrane expression of OATP1B3 in plated OATP1B3-expressing HEK293 cells, OATP2B1 in plated OATP2B1-expressing MDCKII cells, and sodium/taurocholate co-transporting polypeptide (NTCP) in plated NTCP-expressing CHO cells was 63.2% (±1.6%), 37.1% (±15.7%), and 71.7% (±1.2%), respectively. This method of quantifying transporter protein expression in the plasma membrane will be useful in the future to predict transporter-mediated drug disposition.
