The Thermo Scientific Cell Surface Protein Isolation Kit is a complete set of reagents for selective biotinylation and subsequent purification of mammalian cell surface proteins to the exclusion of intracellular proteins.

Features of the Cell Surface Protein Isolation Kit:

• Isolates cell surface proteins—reduces complexity of total cellular protein
• Efficiently recovers labeled proteins—cleavable biotin allows for nearly 100% recovery of isolated cell surface proteins
• Convenience—all reagents are provided in one kit, along with complete instructions for labeling, cell lysis and purification of cell surface membrane proteins
• Western blotting applications—proteins recovered in SDS-PAGE buffer are loaded directly onto polyacrylamide gels
• Robust system—protocol designed for diverse cell lines, including NIH 3T3, HeLa, C6 and A431

The kit efficiently labels proteins that have accessible lysine residues and sufficient extracellular exposure. The isolation procedure uses a cell-impermeable, cleavable biotinylation reagent (Sulfo-NHS-SS-Biotin) to label exposed primary amines of proteins on the surface of intact adherent or suspension cells. Treated cells are then harvested, lysed and the labeled surface proteins are affinity-purified using Thermo Scientific NeutrAvidin Agarose Resin. The isolated cell surface proteins contain a small, nonreactive tag of the originally labeled primary amines but are no longer biotinylated (biotin remains bound to the resin). The kit contains sufficient reagents for eight experiments, each involving four T75 flasks of confluent cells.

The Cell Surface Protein Isolation Kit has been successfully used to label and purify cell surface proteins. In one experiment, HeLa cells were treated with or without Sulfo-NHS-SS-Biotin and the cells were processed according to the kit procedure. Fractions collected, including elution, flow-through and post-elution gel, were analyzed by Western blot. Four cell surface proteins were identified in the elution fraction of cells treated with the biotin label. These proteins included epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor beta (IGF-1β), integrin 1 and integrin 5. In marked contrast, none of these proteins were identified in the elution fraction of unlabeled cells; the unlabeled proteins were exclusively found in the flow-through. Some cell surface proteins were also identified in the flow-through fractions from labeled cells. These proteins were likely not labeled because of steric hindrance. A negligible amount of captured protein was present in the post-elution gel fractions, indicating nearly 100% recovery of captured proteins. A trace amount of heat shock protein 90 (Hsp90) was detected in the elution fraction of all samples tested, demonstrating the specificity of the label for cell surface versus intracellular proteins.

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