Biotinylation and assessment of membrane polarity: caveats and methodological concerns.
Am J Physiol
; 268(2 Pt 2): F285-95, 1995 Feb.
Article
in En
| MEDLINE
| ID: mdl-7864168
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biotin
/
Cell Membrane
/
Cell Polarity
Limits:
Animals
Language:
En
Journal:
Am J Physiol
Year:
1995
Document type:
Article