Biotinylation and Chemical Cross-Linking of Membrane Associated Molecules

Summary

Cell-surface biotinylation is a popular alternative to radioiodination for cell surface labeling in studies aimed at characterizing the molecular interactions between cell surface molecules. However, such studies often require the use of chemical cross-linking agents to cross-link and preserve molecular associations upon cell disruption, and subsequent analysis of specific molecules in the cell lysate by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The procedure described below can be used to simultaneously biotinylate and chemically cross-link lymphocyte surface molecules in a single step. Briefly, the lymphocytes are washed, suspended in phosphate-buffered saline (PBS, pH 8.0), and incubated in the presence of sulfo-NHS-biotin (0.5 mg/ml) and DTSSP (0.2 mg/ml) for 30 min at room temperature. The lymphocytes are then washed and lysed in detergent; the lysates are immunoprecipitated with specific mAbs; and the immunoprecipitates are analyzed by SDS-PAGE and proteins detected by enhanced chemiluminescence (ECL). The biotinylation procedure, with or without chemical cross-linking, can also be used in conjunction with permeabilization of the cells with lysolecithin (15–25μg/ml) to reveal associations with intracellular molecules. The advantage of performing biotinylation and chemical cross-linking on permeabilized cells, rather than in total cell lysates, is that weak molecular associations can be preserved before cell disruption. Also, washing of the permeabilized cells after treatment with reactive biotin and cross-linker avoids the need for removing these reagents from the lysate, since their presence can cause unwanted biotinylation and/or cross-linking of the antibodies used during subsequent immunoprecipitation steps. The procedure employs nonradioactive detection and is particularly useful in studies characterizing the molecular associations of cell surface receptors with other molecules, either on the cell surface or inside the cell.