Plasma membrane biotinylation

NHS esters of D-biotin have been used in many applications, including the biotinylation of rat IgE to study receptors on murine lymphocytes (Lee and Conrad, 1984), in the development of an immunochemical assay for a postsynaptic protein and its receptor (LaRochelle and Froehner, 1986a), in the study of plasma membrane domains by biotinylation of cell surface proteins in Dictyostelium disoideum amoebas (Ingalls et al., 1986), and for the detection of blotted proteins on nitrocellulose membranes after transfer from polyacrylamide electrophoresis gels (LaRochelle and Froehner, 1986b). 

Komiotis D, Wmcel-Drake JD, Dieter JP, Lim CT, LeBreton GC. Labeling of human platelet plasma membrane thromboxaie A/prostqglandin Hj receptors using SQB, a novel biotinylated rec tor probe. Biocfaem Phannacol 1996 52 763-70... 

For shotgnn-LC-MS identification, selective prefractionation by means of AfC is a powerful tool. Biotinylation of cell-surface proteins and subsequent streptavidin-AfC enabled a 1600-fold and 400-fold emichment of plasma membrane proteins over proteins from mitochondria and endoplasmic reticulum, respectively [78-79]. Because of the selective isolation procedure, only 3% of the 918 unique proteins identified were related to mitochondria and endoplasmic reticulum proteins, and 25% were either plasma membrane proteins or membrane-associated proteins. 

One of the biggest advantages of this culture system is the accessibility of either the apical or basolateral surface to any reagent that can be added to the medium in contact with either of the two surfaces. This is the basis of the biotinylation techniques that have been developed to selectively label proteins present in the apical or the basolateral domains of the plasma membrane of filter-grown cells. 

Fig. 1. EFA6 expression promotes the cell surface retention of occludin. The quantity of occludin (A) and transferrin receptor (B) present at the plasma membrane of control and EFA6-expressing MDCK cells was determined after pulse-chase and cell-surface biotinylation. The amounts of protein were quantified by densitometry and plotted against time.

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