• EZ-Link Sulfo-NHS-SS-Biotin, 8 x 6 mg vials, store at 2–8°C
• Lysis Buffer, 10 mL, store at 2–8°C
• NeutrAvidin Agarose, store at 2–8°C
• Wash Buffer, 35 mL, store at 2–8°C
• Elution Buffer, 5 mL, store at 2–8°C
• Column Accessory Pack, 16 spin columns, 16 caps, 16 bottom plugs, and 16 collection tubes, store at room temperature
• Dithiothreitol (DTT), No-Weigh Format, 1 x 7.7 mg microtube, store at 2–8°C
• BupH Phosphate Buffered Saline Pack, 2 packs, store at room temperature
• BupH Tris Buffered Saline Pack, 2 packs, RT

描述

The Thermo Scientific Pierce Cell Surface Protein Biotinylation and Isolation Kit enables the selective biotinylation, solubilization, and enrichment of plasma membrane proteins.

Features of the Cell Surface Protein Biotinylation and Isolation Kit include:
• Improved performance—kit and protocol have been re-designed to improve enrichment and extraction of biotinylated plasma membrane proteins while minimizing intracellular protein contamination
• Optimized—protocol has been refreshed to reduce processing time and the number of steps, and reagent formulations optimized for improved performance
• Compatible—reagents have been verified with suspension and adherent cell lines, with additional instructions provided for MS sample preparation
• Validated applications—reagents and procedures are compatible with western blotting and MS applications

This easy-to-use kit provides all of the necessary components and procedure for optimal labeling and subsequent isolation of cell surface proteins. Buffers are supplied pre-formulated to produce consistent results. Mammalian cells are first labeled with EZ-Link Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent. Cells are subsequently lysed and the labeled proteins are captured with NeutrAvidin Agarose. Dithiothreitol (DTT) is used in the elution to reduce the disulfide bonds in the biotin label, resulting in the release of the bound proteins without the biotin label. This kit contains sufficient reagents for eight experiments, consisting of two 85-95% confluent 15-cm dishes or four 75-cm² flasks.

Cell surface proteins represent a key subset of cellular proteins and play major roles in signal transduction, cell adhesion, and ion transport. These proteins are challenging to efficiently extract and isolate due to their multiple membrane-spanning domains. Often, the harsh detergents and conditions necessary for efficient extraction result in denaturation and contamination of plasma membrane proteins after isolation. As an alternative, this kit utilizes a membrane-impermeable, amine-reactive, sulfo-NHS-SS-biotin to efficiently label cell surface proteins with accessible lysines. The lysis, capture, wash, and elution conditions have been optimized for efficient enrichment and isolation while minimizing contamination of intracellular proteins.

The protocol has been optimized for use with diverse mammalian cell lines, including adherent and suspension cells, and is useful for differential expression analysis between treated and non-treated cells or between one or more cell lines. Selectivity and efficiency of the cell surface protein isolation has been verified by western blotting and LC-MS analysis.

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