Biotinylation and assessment of membrane polarity: Caveats and methodological concerns

Cara J. Gottardi, Lisa A. Dunbar, Michael J. Caplan

Research output: Contribution to journalArticle

168 Scopus citations

Abstract

Studies of epithelial membrane polarity have been greatly facilitated through the use of the N-hydroxysuccinimide-biotin surface labeling technique (M. Sargiacomo, M. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. J. Membr. Biol. 107: 277-286, 1989). We have used this technique in studies on the sorting and targeting of ion-transporting adenosinetriphosphatase molecules in polarized epithelial cells. Through efforts to optimize this technique in our experimental system, we have encountered several experimental conditions and circumstances where biotinylation is extremely inefficient and the assessment of membrane polarity which it provides is misleading. We demonstrate that the pH and ionic strength of the biotinylation buffer can dramatically affect biotin incorporation and that protocol-dependent variations in the recovery of biotinylated proteins can result in misrepresentation of the actual apical/basolateral distribution of a protein. Conditions and protocols that may improve the sensitivity and accuracy of this technique are discussed.

Original languageEnglish (US)
Pages (from-to)F285-F295
JournalAmerican Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume268
Issue number2 37-2
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Physiology

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