Description:
This control plasmid contains a glycosylphosphatidylinositol (GPI) anchor sequence cloned upstream of the ACP-tag coding sequence in pACP. This sequence directs the fusion protein to the plasma membrane with the ACP tag exposed on the outside surface, giving a localized control signal when labeled with ACP-tag substrates. GPI is present in all eukaryotic cells where it anchors glycoproteins to the cell-surface. The GPI anchor helps increase the mobility of membrane proteins, thereby supporting signal transduction and cellular transport. In addition the GPI anchor plays an important role in creating antigens on cell surfaces.
The ACP-tag is a polypeptide tag (8 kDa) based on the acyl carrier protein (ACP). It allows the specific, covalent attachment of virtually any molecule to a protein of interest. ACP-tag substrates are derivates of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue of the ACP-tag by a phosphopantetheine transferase (SFP or ACP Synthase). Having no cysteines, the ACP-tag is particularly suited for specifically labeling cell-surface proteins, and should be useful for labeling secreted proteins with disulphide bridges such as antibodies.
There are two steps to using this system: subcloning and expression of the protein of interest as an ACP-tag fusion, and labeling of the fusion protein with the ACP-tag substrate of choice. The cloning and expression of ACP-tag protein fusions is described in documents provided with the pACP cloning plasmid. The labeling of fusion proteins with ACP-tag substrates is described in the documentation supplied with ACP-tag substrates and ACP or SFP Synthase.
Materials Required but not Supplied:
Mammalian cell lines
Transfection reagents
ACP-tag substrates
SFP or ACP Synthase
Live CHOK1 cells transiently transfected with pACP-GPI. Cells were labeled with CoA 488 using ACP Synthase for 30 minutes and imaged by confocal microscopy.
Reaction & Storage Conditions
Storage Temperature:
-20°C
Notes
General notes:- Storage: pACP-GPI is supplied in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at a concentration of 0.5 µg/µl. Plasmid solutions can be stored at 4°C for up to one week. For long-term storage -20°C is recommended.
Protocols
Protocols for pACP-GPI Control Plasmid
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road Ipswich, MA 01938. For detailed information, see www.neb.com/cia/legal/.
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