Isolate cell surface proteins through biotin-labeling and affinity selection.

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.

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 and 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.

Highlights:

• 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

Product Details:

Protocol summary for the Thermo Scientific Pierce Cell Surface Protein Isolation Kit. The kit provides a convenient and efficient method to isolate and purify the cell surface protein fraction of cultured mammalian cells. Recovered proteins are immediately ready for Western blot analysis.

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.

Specific extraction and isolation of cell surface proteins. Panels are Western blot results for known cell surface proteins (Panel A) and intracellular proteins (Panel B) from HeLa cells processed using the Thermo Scientific Cell Surface Protein Isolation Kit. Plus symbol (+) denotes results for cells treated with the Sulfo-NHS-SS-Biotin reagent; minus symbol (-) denotes results for cells that were not treated with the biotin reagent but were otherwise carried through the kit procedure. Lanes are no-sample resin control (R), flow-through (F) and eluted (E) fractions. Presence of target cell surface proteins in the plus-E and minus-F conditions indicate successful isolation with the kit. Presence of intracellular proteins in F condition of both plus and minus conditions indicates that the labeling and purification procedure is specific to cell surface proteins.

Differential expression of cell surface proteins in response to EGF. A431 and HeLa cells were treated with or without 20ng/mL and 10ng/mL EGF for 16 hours, respectively. Both cell types were processed with the Thermo Scientific Pierce Cell Surface Protein Isolation Kit protocol. Elution fractions were analyzed by Western blot for the quantities of A. integrin β1 and integrin α5 subunits or B. EGFR.

Validation of protocol with multiple cell lines. NIH3T3, C6 and HeLa cells were labeled with Sulfo-NHS-SS-Biotin and triplicate samples were processed as described in Methods. Flow-through and elution fractions were analyzed by Western blot for cell surface proteins or Hsp90, an intracellular protein. A. NIH3T3, B. C6 and C. HeLa. Legend: (F) flow-through and (E) elution.

Related Products:

Pierce Cell Lysis Reagents
DTT (dithiothreitol) microtubes
EZ-Link Sulfo-NHS-SS-Biotin
Biotin Quantitation Kit
NeutrAvidin Agarose Resin
Empty Spin Columns

References

1. Handlogten, M.E., et al. (2005) Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3). Am J Physiol Renal Physiol.289, F347-F358.
2. Ding, S., et al. (2005) Investigating the Putative Glycine Hinge in Shaker Potassium Channel. J. Gen. Physiol.126, 213-226.