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Cell Surface Biotinylation Protocol

Biotinylation uses the ability of the soluble vitamin biotin to bind to cell proteins. Biotin binds to the protein strepavidin and is, therefore, useful in cell biology to visualize cell proteins. Biotinylation allows the safe labeling of proteins compared to techniques such as radioactivity and does not affect the cell's normal function. It allows researchers to study cell proteins such as receptors, and that enables a greater understanding of diseases and disease processes.

  1. Labeling Suspended Cells

    • Wash cells in ice-cold phosphate buffered saline (PBS) to remove contaminants. Buffers containing tris and glycine will compete with biotinylation and should be avoided. To avoid biotinylating internal proteins, the labeling can be performed at 4 degrees Celsius or in the presence of azide. Resuspend cells in PBS and the soluble biotin, sulfo-NHS-LC-biotin.

    Labeling Adherent Cells

    • Wash cells in PBS and incubate with a product such as sulfo-NHS-LC-biotin. The cells are then scraped into a dish and lysed in a buffer containing the detergent Triton-X 100 and prepared for visualization.

    Preparing Labeled Cells

    • Following incubation, the cells are centrifuged into a pellet and washed in PBS. The cells are resuspended in PBS and purified with the IgG sepharose gel. The bound protein is diluted in loading buffer and heated to 100 degrees Celsius.

    Visualizing Cells

    • Diluted proteins can be visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting with the protein and enzyme mix, streptavidin-horseradish peroxide. Blots are visualized with a chemical reaction --- chemiluminescence --- which emits light. Specificity of the biotinylation can be confirmed with antibodies for the protein under study.

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