Cellular Analysis
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  • Cellular Analysis

    SNAP- and CLIP-tag protein labeling systems enable the specific, covalent attachment of virtually any molecule to a protein of interest. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag® fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag substrates are dyes, fluorophores, biotin, or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag™ is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells. 

    SNAP-tag® is a registered trademark of New England Biolabs, Inc.
    CLIP-tag™ is a trademark of New England Biolabs, Inc.

    • Fluorescent Labeling of COS-7 Expressing SNAP-tag® Fusion Proteins for Live Cell Imaging

      Watch as Chris Provost, of New England Biolabs, performs fluorescent imaging of live COS-7 cells expressing SNAP-tag® fusion proteins.

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    • SNAP-tag® Overview Tutorial

      View an interactive tutorial explaining the mechanism of our SNAP-tag® technologies and reagents available for researchers wishing to study the function and localization of proteins in live or fixed cells.

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    Cellular Analysis includes these subcategories:

    Starter Kits Information
    SNAP-tag® Substrates Information
    CLIP-tag™ Substrates Information
    ACP/MCP-tag Substrates Information
    SNAP-Capture
    Blocking Agents Information
    Cloning Vectors & Control Plasmids Information
    Synthases Information
    Biotin/Vista Labels Information
    Building Blocks Information

      Publications related to Cellular Analysis:

    1. Damoiseaux, R. et al (2002) Towards the generation of artificial O6-alkylguanine-DNA alkyltransferases: in vitro selection of antibodies with reactive cysteine residues ChemBioChem 3, 573-575. PubMedID: 12325014
    2. Keppler A. et al. (2003)A general method for the covalent labeling of fusion proteins with small molecules in vivo Nature Biotechnology 21, 86-89.
    3. Gendreizig S. et al. (2003)Covalent labeling of fusion proteins with chemical probes in living cells Chimia 57, 181-183.
    4. Juillerat A. et al. (2003)Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo Chem. Biol.  10, 313-317.
    5. Gendreizig, S. et al. (2003)Induced protein dimerizaton in vivo through covalent labeling JACS 125, 14970-14971. PubMedID: 14653715
    6. Keppler A. et al. (2004)Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vitro and in vivo Methods 32, 437-444. PubMedID: 15003606
    7. Keppler A. et al. (2004)Labeling of fusion proteins with synthetic fluorophores in live cells PNAS 101, 9955-9959.
    8. Kindermann M. et al. (2004)Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins Bioorg. Med. Chem. Lett. 14, 2725-2728.
    9. Juillerat A. et al. (2005)Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells ChemBioChem 6, 1263-1269. PubMedID: 15934048
    10. Johnsson N. et al. (2005)Protein chemistry on the surface of living cells Chembiochem. 6, 47-52. PubMedID: 15558647
    11. Regoes A. et al. (2005)SNAP-tag mediated live cell labeling as an alternative to GFP in anaerobic organisms BioTechniques 39, 809-812.
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    14. Tirat A. et al. (2006)Evaluation of two novel tag-based labeling technologies for site-specific modification of proteins Int. J. Biol. Macromol. 39, 66-76. PubMedID: 16503347
    15. Heinis C. et al. (2006)Evolving the substrate specificity of O6 alkylguanine DNA alkyltransferase through loop insertion for applications in molecular imaging ACS Chem Biol. 1, 575-584. PubMedID: 17168553
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    39. McMurray, M.A. and Thorner, J. (2008)Septin stability and recycling during dynamic structural transitions in cell division and development Current Biology 18, 1203-1208. PubMedID: 18701287
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    41. Hill Z. B. (2009)A chemical genetic method for generating bivalent inhibitors of protein kinases J. Am. Chem. Soc. 131, 6686-6688. PubMedID: 19391594
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    44. Sletten E. and Bertozzi C. (2009)Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality Angew. Chem. Int. Ed. 48, 6974-6998. PubMedID: 19714693
    45. Carroll C.W. et al. (2009) Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N Nat. Cell Biol. 11, 896-902. PubMedID: 19543270
    46. Foltz D.R. et al. (2009)Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP Cell 137, 472-84. PubMedID: 19410544
    47. Donovan C. et al. (2009)Characterization and subcellular localization of bacterial flotillin homologue Microbiology 155, 1786-1799. PubMedID: 19383680
    48. Keppler A. et al. (2009)Chromophore-assisted laser inactivation of α- and γ-tubulin SNAP-tag fusion proteins inside living cells ACS Chem. Biol. 4, 127-138. PubMedID: 19191588
    49. Chattopadhaya S. et al. (2009)Expanding the chemical Biologist's tool kit: chemical labelling strategies and its applications Curr. Med. Chem.  16, 4527-4543. PubMedID: 19903152
    50. Cornish, V. W. (2009)Fluorescence in living systems: applications in chemical biology Wiley Encyc. of Chem. Biol. 2, 28-38.
    51. Degorce F. et al. (2009)HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications Curr. Chem. Genomics 3, 22-32. PubMedID: 20161833
    52. Samoshkin A. et al. (2009)Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation PLoS One 4, e6831. PubMedID: 19714251
    53. Böhme I and Beck-Sickinger A. G. (2009)Illuminating the life of GPCRs Cell Commun. Signal 7, 16. PubMedID: 19602276
    54. Bannwarth et. al. (2009)Indo-1 Derivatives for local calcium sensing JACS Chemical Biology 4, 179-190. PubMedID: 19193035
    55. Milenkovic L. et al. (2009)Lateral transport of smoothened from the plasma membrane to the membrane of the cilium J. Cell Biol. 187, 365-374. PubMedID: 19193035
    56. Farr G. A. et al. (2009)Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells J. Cell Biol. 186, 269-282. PubMedID: 19620635
    57. Tivari R. and Parang K. (2009)Protein conjugates of SH3-domain ligands and ATP- competitive inhibitors as bivalent inhibitors of protein kinases ChemBioChem. 10, 2445 - 2448. PubMedID: 19731277
    58. Brun M.A. et al. (2009)Semisynthetic fluorescent sensor proteins based on self-labeling protein tags J. Am. Chem. Soc. 131, 5873-5784. PubMedID: 19348459
    59. Kapmeier F. et al. (2009)Site-Specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase Bioconjug Chem. 23-Apr, PubMedID: 19388673
    60. Uano Y. and Matsuzaki K. (2009)Tag-probe labeling methods for live-cell imaging of membrane proteins Biochim. Biophys. Acta. 1788, 2124-2131. PubMedID: 19646952
    61. Johnsson K. (2009)Visualizing biochemical activities in living cells Nat Chem Biol 5, 63-65. PubMedID: 19148167
    62. Nicolle O. et al. (2010)Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis FEMS Immunol. Med. Microbiol.  59, 357-363. PubMedID: 20482622
    63. Ruggiu A. A. et al. (2010)Fura-2FF-based calcium indicator for protein labeling Org. Biomol. Chem. 8, 3398-3401. PubMedID: 20556282
    64. Campos, C. et al. (2010)Labeling cell structures and tracking cell lineage in zebrafish using SNAP-Tag Dev. Dynamics 240, 820-827. PubMedID: 21360787
    65. Alvarez-Curto J. et al. (2010)Ligand regulation of the quaternary organization of cell surface M3 muscarinic acetylcholine receptors analyzed by fluorescence resonance energy transfer (FRET) imaging and homogenous time-resolved FRET J. Biol. Chem. 285, 23318-23330. PubMedID: 20489201
    66. Ciruela F. et al. (2010)Lighting up multiprotein complexes: lessons from GPCR oligomerization Trends Biotechnol 28, 407-415. PubMedID: 20542584
    67. Kamiya M. and Johnsson K. (2010)Localizable and Highly Sensitive Calcium Indicator Based on a BODIPY Fluorophore Anal. Chem. 82, 6472-6479. PubMedID: 20590099
    68. Rhee S. G. et al. (2010)Methods for detection and measurement of hydrogen peroxide inside and outside of cells Mol. Cells 29, 539-549. PubMedID: 20526816
    69. Srikun, D. et al. (2010)Organelle-targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP-tag protein labeling  J. Am. Chem. Soc. 132, 4455-4465. PubMedID: 20201528
    70. Maurel D. et al. (2010)Photoactivatable and photoconvertible fluorescent probes for protein labeling ACS Chem. Biol. Asap PubMedID: 20218675
    71. Kampmeier, F. et al. (2010)Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein Eur. J. Med. Mol. Imaging PubMedID: 20449589, DOI: 10.007/S00259-010-1482-5
    72. Hein B. et al. (2010)Stimulated emission depletion nanoscopy of living cells using SNAP-Tag fusion proteins Biophys. J.  98, 158-163. PubMedID: 20074516
    73. Dellagiacoma, C. et al. (2010)Targeted photoswitchable probe for nanoscopy of biological structures ChemBioChem PubMedID: 20540058, DOI: 10.1002/Cbic.201000189
    74. Geissbuehler M. et al. (2010)Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell Biophys. J. 98, 339-349. PubMedID: 22259112
    75. Eckhardt, M. et al. (2011)A SNAP-tagged detivative of HIV-1 - A versatile tool to study virus-cell interactions PLoS One 6:e22007. PubMedID: 21799764, DOI: 10.137/journal. P One .0022007
    76. Hoskins, A. et al. (2011)Ordered and dynamic assembly of single spliceoseoms Science 331, 1289. PubMedID: 21393538
    77. Kindermann M. et al. (2003)Covalent and selective immobilization of fusion proteins JACS 125, 7810-7811. PubMedID: 12822993
    78. La Clair, J.J. et al. (2004)Manipulation of carrier proteins in antibiotic biosynthesis Chem. Biol. 11, 195-201. PubMedID: 15123281
    79. George N. et al. (2004)Specific labeling of cell surface proteins with chemically diverse compounds J .Am. Chem. Soc.  126, 8896-8897. PubMedID: 15264811
    80. Huber W. et al. (2004)SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins Anal. Biochem. 333, 280-288. PubMedID: 15450803
    81. Kufer S.K. et al. (2005)Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy Eur. Biophys. J 35, 72-78. PubMedID: 16160825
    82. Yin J. et al. (2005)Labeling proteins with small molecules by site-specific posttranslational modification J Am Chem Soc 126, 7754-7755. PubMedID: 15212504
    83. Cravatt B.F. (2005)Live chemical reports from the cell surface Chem. Biol. 12, 954-956. PubMedID: 16183017
    84. Vivero-Pol L. et al. (2005)Multicolor imaging of cell surface proteins J. Am. Chem. Soc. 127, 12770-12771. PubMedID: 16159249
    85. Tugulu S. et al. (2005)Protein-functionalized polymer brushes Biomacromolecules 6, 1602-1607. PubMedID: 15877383
    86. Yin J. et al. (2005)Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling Chem. Biol 12, 999-1006. PubMedID: 16183024
    87. Meyer B.H. et al. (2006)Covalent labeling of cell-surface proteins for in vivo FRET studies FEBS Letters 580, 1654-1658. PubMedID: 16497304
    88. Meyer B.H. et al. (2006)FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells Proc. Natl. Acad. Sci. USA 103, 2138-43. PubMedID: 16461466
    89. Prummer M. et al. (2006)Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells ChemBioChem 7, 908-911. PubMedID: 16607667
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    91. Jongsma M.A., Litjens R. H. (2006) Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address  Proteomics 6, 2650-2655. PubMedID: 16596705
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    93. Zhou Z. et al. (2007)Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases ACS Chemical Biology 2, 337-346. PubMedID: 17465518
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    Applications

    • Simultaneous dual protein labeling inside live cells
    • Protein localization and translocation
    • Pulse-chase experiments
    • Receptor internalization studies
    • Selective cell surface labeling
    • Protein pull-down assays
    • Protein detection in SDS-PAGE
    • Flow cytometry
    • High throughput binding assays in microtiter plates
    • Biosensor interaction experiments
    • FRET-based binding assays
    • Single molecule labeling
    • Super-resolution microscopy

    Features

    • Clone and express once, then use with a variety of substrates
    • Non-toxic to living cells
    • Wide selection of fluorescent substrates
    • Highly specific covalent labeling
    • Simultaneous dual labeling

    Protein Labeling with SNAP-tag and CLIP-tag

    The SNAP- (gold) or CLIP-tag (purple) is fused to the protein of interest (blue). Labeling occurs through covalent attachment to the tag, releasing either a guanine or a cytosine moiety.

    SNAP-tag®, CLIP-tag™ and ACP/MCP-tag Substrate Selection Chart

    NEB offers a large selection of fluorescent labels (substrates) for SNAP-, CLIP-, ACP- and MCP-tag fusion proteins.