Pierce Cell Surface
Protein Isolation Kit

The Pierce Cell Surface Protein Isolation Kit is a complete kit for the convenient biotinylation and purification of mammalian cell surface proteins. Adherent or suspended cells are first labeled with Sulfo-NHS-SS-Biotin, a cleavable biotinylation reagent. Efficient labeling requires accessible lysine residues, sufficient sequence with extracellular exposure and minimal steric hindrance. The cells are subsequently lysed with a mild detergent and then labeled proteins are isolated with immobilized NeutrAvidin Gel. The bound proteins are released by incubating the resin with SDS-PAGE sample buffer containing 50 mM DTT. The reducing agent cleaves the disulfide bond within the biotin label, and nearly 100% of the bound proteins are recovered. The protocol is optimized for diverse cell lines, including NIH3T3, HeLa and C6. Isolated proteins can be analyzed by Western blot, allowing for differential expression analysis between treated and nontreated cells or between two or more cell lines.

Highlights:

• Isolation of cell surface proteins - reduces complexity of total cellular protein
• Efficient recovery of labeled proteins - nearly 100% recovery of isolated cell surface proteins from NeutrAvidin Gel
• Convenient purification - all reagents are provided in the kit with complete instructions for labeling, cell lysis and purification
• 1-D Western blotting applications - proteins recovered in SDS-PAGE sample buffer are loaded directly onto SDS-polyacrylamide gels
• Robust system - protocol designed for diverse cell lines

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Figure 1. Procedure for the Cell Surface Protein Isolation Kit.

The Pierce Cell Surface Protein Isolation Kit was successfully used to label and purify cell surface proteins. HeLa cells were treated with or without Sulfo-NHS-SS-Biotin and the cells were processed as described in Figure 1. 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 (Figure 2). 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.

Figure 2. The Pierce Cell Surface Protein Isolation Kit specifically purifies proteins localized to the cell surface. HeLa cells were treated or not treated with Sulfo-NHS-SS-Biotin and processed according to the kit protocol. Elution fractions, post-elution gel and flow-through were analyzed by Western blot for A. cell surface proteins, including EGFR, IGF-1 , integrin 1 and integrin 5 and B. heat shock protein 90 and calnexin, two intracellular proteins. Legend: (+) label,
(-) no label, (R) gel, (F) flow-through and (E) elution.

Figure 3. Differential expression of cell surface proteins in response to EGF.
A431 and HeLa cells were treated with or without 20 ng/ml and 10 ng/ml EGF for 16 hours, respectively. Both cell types were processed with the 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.

Figure 4. 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.

The Pierce Cell Surface Protein Isolation Kit provides a convenient and efficient method for the isolation of cell surface proteins from cultured mammalian cells. Recovered proteins are immediately ready for Western blot analysis.

Poppers Cell Lysis Reagents		No-Weigh DTT
EZ-Link Sulfo-NHS-SS-Biotin		EZ Biotin Quantitation Kit
Immobilized NeutrAvidin Protein		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.