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pMCP-GPI Control Plasmid
This product was discontinued on 01/01/2013
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Properties and Usage
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Description
This control plasmid contains a glycosylphosphatidylinositol (GPI) anchor sequence cloned upstream of the MCP-tag coding sequence in
pMCP-tag(m). This sequence directs the fusion protein to the plasma membrane with the MCP-tag exposed on the outside surface, giving a
localized control signal when labeled with CoA 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 MCP-tag is a small protein tag (8 kDa) based on the acyl carrier protein (ACP) containing two mutations (D36-T36 and D39-G39), for
labeling cell membrane proteins. It allows the specific, covalent attachment of virtually any molecule to a protein of interest. MCP-tag
substrates are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently
attached to a conserved serine residue of the MCP-tag by a phosphopantetheine transferase (SFP Synthase). Having no cysteines, the
MCP-tag is particularly suited for specifically labeling cell surface proteins, and should be useful for labeling secreted proteins with disulfide
bridges such as antibodies.
There are two steps to using this system: sub cloning and expression of the protein of interest as an MCP-tag fusion, and labeling of the
fusion protein using SFP Synthase with the substrate of choice.The cloning and expression of MCP-tag protein fusions is described in
documents provided with the pMCP-tag(m) cloning plasmid. The labeling of fusion proteins with CoA substrates is described in the
documentation supplied with CoA substrates and SFP Synthase.
Live CHO-K1 cells transiently transfected with pMCP-GPI. Cells were labeled with CoA 488 (green) using SFP Synthase for 30 minutes.
Properties and Usage
Materials Required but not Supplied
Mammalian cell lines
Transfection reagents
CoA substrates
SFP Synthase
Storage Temperature
-20°C
Sequence Files
Fasta GenBank
Related Products
Companion Products
NEB® 10-beta Competent E. coli (High Efficiency)
Notes
1. pMCP-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.
2. NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating this control plasmid.
FAQs
Tech Tips
FAQs
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What is the ACP-tag?
How does it work?
How specific is the binding of substrate to the ACP-tag?
What linker type and length would you recommend?
Can I clone my protein as fusion to the N- or C-terminus of the ACP-tag?
Are ACP-tag substrates stable to fixation?
Can ACP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
Can you use ACP-tag for in vivo FRET?
Does the ACP-tag labeling reaction work in Yeast?
Tech Tips
If you generate more plasmid DNA by bacterial transformation, we recommend isolating the plasmid DNA using an endotoxin-free
plasmid prep kit prior to transfection into mammalian cells.
Protocols
Datacards
Protocols
1. Expression of MCP-GPI (N9320)
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and
quality controls. The following file naming structure is used to name the majority of these document files: [Catalog
Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the
Technical Support Form for appropriate document.
N9320Datasheet-Lot0011209
Selection Charts
Interactive Tools
Troubleshooting Guides
Selection Charts
Comparison of SNAP-tag®/CLIP-tag™ Technologies to GFP
SNAP-tag®/CLIP-tag® Cloning Vector and Plasmid Selection Chart
Troubleshooting Guides
Labeling with SNAP-tag® Technology Troubleshooting Guide
Interactive Tools
DNA Sequences and Maps Tool
NEBioCalculator
Safety Data Sheet
Datacards
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
pMCP-GPI Control Plasmid
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and
quality controls. The following file naming structure is used to name the majority of these document files: [Catalog
Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the
Technical Support Form for appropriate document.
N9320Datasheet-Lot0011209
Legal and Disclaimers
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc
(NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes
in humans or animals.
Licenses
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.
Cellular Imaging and Analysis (i.e., SNAP and CLIP products)
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH
AND DEVELOPMENT ONLY. Commercial use of this system or any components thereof requires a license from New England
Biolabs, Inc., 240 County Road, Ipswich, MA 01938.
The products and/or their use may be covered by one or more of the following patents and patent applications:
7,939,284 (Methods for Using O6-Alkylguanine-DNA-Alkyltransferases)
7,888,090 (Mutants of O6-Alkylguanine-DNA-Alkyltransferases)
8,163,479 Specific Substrates for O6-Alkylguanine-DNA-Alkyltransferases)
8,178,314 (Pyrimidines Reacting With O6-Alkylguanine-DNA-Alkyltransferases)
PCT/EP2007/057597 (Labeling of Fusion Proteins with Synthetic Probes)
EP07117800 (Drug Delivery)
EP07117802 (Drug Delivery)
EP07120288 (GTPase-Transient Protein Protein Interactions)
These patents and patent applications are owned by Covalys, or owned by the Ecole Polytechnique Fédérale de Lausanne (EPFL)
and exclusively licensed to Covalys and NEB.
The CMV promoter is covered under U.S. Patent No. 5,385,839 and its use is permitted for research purposes only. Any other use
of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa
City, IA 52242.