This control plasmid contains the gene encoding the Beta-2 adrenergic receptor cloned downstream of the SNAPf coding sequence in pSNAPf as a fusion to the C-terminus of the SNAP-tag. The signal peptide fused to the N-terminus of SNAPf is based on the 5HT3A serotonin receptor. The Beta-2 adrenergic receptor is a member of the G protein coupled receptors and mediates the catecholamine-induced activation of adenylate cyclase through the action of G proteins.
The SNAPf-Beta-2 adrenergic receptor is inserted in the plasma membrane with the SNAPf exposed to the extracellular side of the membrane. When labeled with SNAP-tag substrates, it gives a selective cell membrane fluorescent labeling pattern.
The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT). SNAP-tag substrates are derivatives of benzylguanines and benzylchloropyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
pSNAPf-ADRβ2 contains an improved version of SNAP-tag, termed SNAPf. SNAPf displays faster kinetics in in vitro labeling and fast, specific and efficient labeling in live and fixed cell applications, thereby rendering it a desired research tool for analysis of protein dynamics.
There are two steps to using this system: sub cloning and expression of the protein of interest as a SNAPf fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAPf-ADRβ2 fusion protein is described in this document. The labeling of the fusion proteins with SNAP-tag substrates is described in the instructions supplied with SNAP-tag substrates.
DNASU is a central repository for plasmid clones and collections that may also be helpful.
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