G-Biosciences' HOOK™ Cell Surface Protein Isolation kit uses our HOOK™ biotin labeling and purification technology in conjunction with our Mammalian Cell PE LB™ lysis buffer to conveniently label cell surface proteins and isolate them for further analysis, including Western blotting

Mammalian cells, adherent or non-adherent, are first labeled with HOOK™ Sulfo-NHS-SS-Biotin, a water-soluble, amine reactive biotinylation reagent that has a sulfo-NHS-ester. The addition of a charged sulfonate (SO₃¯) on the N-hydroxysuccinimide ring of the sulfo-NHS ester results in its solubility in water, but prevents it permeating plasma membranes. The solubility and impermeability to plasma membranes makes HOOK™ Sulfo-NHS-SS-Biotin ideal for studying cell surface proteins as it will only react with the protein molecules on the outer surface of plasma membranes. An additional advantage of HOOK™ Sulfo-NHS-SS-Biotin is the presence of a disulfide bond in the spacer arm. The disulfide bond permits the cleavage of the biotin moiety from the protein, making its interaction with avidin/ streptavidin reversible.

Following labeling, cells are lysed with G-Biosciences' Mammalian Cell PE LB™, a buffered lysis solution that employs a mild non-ionic detergent for enhanced extraction and stability of proteins, and the cell lysate is applied to a Streptavidin agarose column. Unlabeled intracellular proteins are washed away and the biotin labeled cell surface proteins are then released by reduction of the disulfide bond with DTT.

G-Biosciences' HOOK™ Cell Surface Protein Isolation kit is supplied with all the necessary reagents and buffers for convenience and improved reproducibility. The kit is compatible with a wide variety of mammalian cells and can be used to compare treated and untreated cells and differences between different cell lines. This kit is supplied with sufficient reagents for five experiments, with each experiment consisting of four 90-95% confluent T-75 cm² flasks.