HOOK™ Cell Surface Protein Isolation kit uses G-Biosciences 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. HOOK
™ Sulfo-NHS-SS-Biotin is a water-soluble, amine reactive biotinylation reagent that has a N-hydroxysulfosuccinimide (sulfo-NHS) ester (see table). The addition of a charged sulfonate (SO
3-) on the N-hydroxysuccinimide ring of the sulfo-NHS esters 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 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.
HOOK™ sulfo-NHS-SS-Biotin
MOLECULAR WEIGHT: 606.69
SPACER ARM (Å): 24.3
REACTIVE GROUP: sulfo-NHS-ester
MEMBRANE PERMEABLE: NO
WATER SOLUBLE: YES
CLEAVABLE/ REVERSIBLE: YES
REACTIVE pH: 7-9
Following labeling, cells are lysed with 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.
The 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-75cm2flasks.
Features
- Complete cell surface biotin labeling and isolation.
- Suitable for a wide selection of mammalian cells.
Applications
- For the isolation of cell surface proteins.
- For the study of cell surface trafficking.
- For the study of receptor:ligand interactions.