Biotinylation cell surfaces

The isolated cells may be lysed using standard mechanical or detergent methods and the biotinylated cell-surface proteins analyzed or isolated using (strept)avidin reagents. 

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

NHS-LC-biotin can be used to add a biotin tag to monoclonal antibodies directed at certain tumor antigens. The biotinylated monoclonals are allowed to bind to the tumor cell surfaces in vivo, and subsequent administration of an avidin or streptavidin conjugate can form the basis for inducing cytotoxic effects or creating traceable complexes for use in imaging techniques (Hnatowich et al., 1987). 

The following protocol is based on the method of Thermo Fisher, as found in the instructions for the cell-surface biotinylation kit. 

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

Schuberth, H.-J. et al. (1996) Biotinylation of cell surface MHC molecules A complementary tool for the study of MHC class II polymorphism in cattle. J. Immunol. Meth. 189, 89-98. 

Polythiophenes functionalized with monosaccharides have been evaluated for their ability to detect the influenza virus and E. coli (Baek et al. 2000). Copolymers of thiophene acetic acid 10 and carbohydrate-modified thiophenes 11 have been prepared via iron(III) chloride mediated polymerization. Addition of influenza virus to a sialic acid containing copolymer resulted in a blue shift of the polymer absorption maximum, resulting in an orange to red chromatic transition. Mannose-containing polythiophenes underwent color changes upon the addition of the lectin ConA or E. coli cells that contain cell surface mannose-binding receptors. A similar biotinylated pol5hhiophene afforded a streptavidin responsive material (Paid and Leclerc 1996). 

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). 

Parrott,M.B. andBarryM. A. (2001) Metabolic biotinylation of secreted and cell surface proteins from mammalian cells. Biochem. Biophys. Res. Commun. 281, 993-1000... 

An avidin-biotin system has been used to attach antibodies in the bilayer of DDSs. Xiao et al. developed a three-step strategy to improve the tumor-to-tissue ratio of anticancer agents [184], Two antibodies specific for the CA-125 antigen that is highly expressed on NIH OVCAR-3 cells were used. These cells were prelabeled with biotinylated anti-CA-125 antibody and fluoroscein isothiocyanate (FITC)-labeled streptavidin (SAv) prior to administration of biotinylated liposomes. Both antibodies were specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells, which do not express the specific antibody. Antibody biotinylation did not affect its immunoreactivity. 

Palenik, B., and Koke, J. A. (1995). Characterization of a nitrogen-regulated protein identified by cell surface biotinylation ofa marine phytoplankton. Appl. Environ. Microbiol. 61, 3311—3315. 

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. 

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