Biotinylation and assessment of membrane polarity: caveats and methodological concerns.
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.
Gottardi
C J
CJ
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Dunbar
L A
LA
Caplan
M J
MJ
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D002021:Buffers; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D016764:Cell Polarity; D004285:Dogs; D004492:Edetic Acid / Q000494:pharmacology; D005374:Filtration; D006388:Hemagglutinins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D018374:LLC-PK1 Cells / Q000378:metabolism; D008722:Methods; D011233:Precipitin Tests; D012685:Sepharose; D019809:Streptavidin; D013552:Swine
D001426:Bacterial Proteins; D002021:Buffers; D006388:Hemagglutinins; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin; D004492:Edetic Acid