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New England Biolabs Cell-Imaging Plasmid Collection

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New England Biolabs offers non-toxic protein labeling systems based on SNAP-, CLIP-, ACP-, and MCP-tags for visualization of proteins inside living or fixed cells. The plasmids associated with this technology are available at Addgene for distribution to the research community.

Key features:

  • Allows for visualization of multiple proteins simultaneously with dual labeling when used with two substrates
  • Can be used in live or fixed cells, and for internal or surface proteins
  • Single constructs are compatible with multiple applications (different fluorophores, biotin, bead purification, blocking agents)
  • ACP- and MCP-tags are used exclusively for surface proteins and use enzymatic binding to attach substrate
  • Control plasmids with a recognizable sub-cellular localization (membrane, nucleus, etc.) are also available
  • Non-toxic to living cells

To learn more about this system, visit the New England Biolabs website. For a comprehensive comparison to GFP, please refer to NEB's comparison of SNAP-tag, CLIP-tag, and GFP.

Technology

Diagram of a SNAP tag reaction. A protein of interest fused to a SNAP tag reacts with a benzylguanine fluorophore to make a stable adduct with guanine leftover.

SNAP-tag

SNAP-tag is based on the human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein, and is suitable for both cell-permeant and non-cell-permeant proteins.

Diagram of a CLIP tag reaction. A protein of interest fused to a CLIP tag reacts with a benzylcytosine fluorophore to make a stable adduct with cytosine leftover.

CLIP-tag

CLIP-tag is identical in function to SNAP-tag but reacts with benzylcytosine rather than benzylguanine derivatives to allow for dual labeling and visualization of complementary protein function or localization.

Diagram of an ACP or MCP tag reaction. A protein of interest fused to an ACP or MCP tag reacts with CoA fused to a label in the presence of ACP or SFP synthase to produce a labeled ACP or MCP tag fusion protein adduct.

ACP and MCP tags

ACP- and MCP-tags are small protein tags based on the acyl carrier protein. Substrates are derivatives of Coenzyme A (CoA). These tags are particularly well suited for labeling cell-surface proteins, and are useful for labeling secreted proteins with disulfide bridges such as antibodies. MCP-tag is a mutant of ACP-tag, and is labeled by SFP Synthase but not by ACP Synthase, thereby permitting selective simultaneous labeling in a sample. Please see the note below for information on substrates and synthases needed for use of these tags.

Plasmids

Addgene ID Plasmid Substrate Feature
101137 pSNAP-tag (T7) Vector SNAP-tag Empty backbone for bacterial expression under T7 control
101135 pSNAP-tag (m) Vector SNAP-tag Empty backbone for stable and transient mammalian expression
101133 pSNAP-CaaX Control Plasmid SNAP-tag Control for endomembrane (CaaX box) localization
101129 pSNAPf-Cox8A Control Plasmid SNAP-tag Control for mitochondrial localization
101124 pSNAPf-H2B Control Plasmid SNAP-tag Control for nuclear localization
101123 pSNAPf-ADRβ2 Control Plasmid SNAP-tag Control for cell surface localization
101136 pCLIP-tag (m) Vector CLIP-tag Empty backbone for stable and transient mammalian expression
101134 pCLIPf-H2B Control Plasmid CLIP-tag Control for nuclear localization
101130 pCLIPf-Cox8A Control Plasmid CLIP-tag Control for mitochondrial localization
101125 pCLIPf-NK1R Control Plasmid CLIP-tag Control for cell surface localization
101127 pMCP-tag (m) Vector ACP/MCP-tag Empty backbone for transient mammalian expression
101126 pACP-tag (m)- 2 Vector ACP/MCP-tag Empty backbone for stable and transient mammalian expression
101132 pMCP-GPI Control Plasmid ACP/MCP-tag Control for cell surface localization
101131 pACP-GPI Control Plasmid ACP/MCP-tag Control for cell surface localization
101128 pACP-ADRβ2 Control Plasmid ACP/MCP-tag Control for cell surface localization

Supplemental Information

SNAP and CLIP tags

ACP and MCP tags

  • ACP-MCP-Tag plasmids - Substrate source (PDF, 46 KB)
  • Please note: New England Biolabs (NEB) has discontinued the ACP-tag/MCP-tag product line from their product portfolio, including the CoA-derivative substrates and the ACP and SFP Synthases. Users may still generate their own fluorescent or non-fluorescent CoA substrates by purchasing coenzyme A disodium salt from a commercial supplier and conjugating it to the label of their choice according to the methods described by George et al. (2004). The ACP and SFP Synthases (AcpS and Sfp, respectively) may be expressed and purified as described previously in Stachelhaus et al. (1998)

References

  • George N, Pick H, Vogel H, Johnsson N, Johnsson K. 2004. Specific labeling of cell surface proteins with chemically diverse compounds. J Am Chem Soc. 126(29):8896-7. PMID:15264811
  • Stachelhaus T, Mootz HD, Bergendahl V, Marahiel MA.1998. Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J Biol Chem. 273(35):22773-81. PMID:9712910
  • Vivero-Pol L, George N, Krumm H, Johnsson K, Johnsson N. 2005. Multicolor imaging of cell surface proteins. J Am Chem Soc. 127(37):12770-1. PMID:16159249