Cellular Analysis
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  • Protein Localization

    Protein functional activities correspond with their subcellular expression and molecular complexing interactions. Localization can be effectively demonstrated with fluorescence microscopy based techniques or fractionation procedures. A broad spectrum of fluorescence imaging can be accomplished by using recombinant reporter proteins, (i.e. GFP, SNAP-tag®), or fluorescent dyes (i.e. Alexa), or fluorophore labeled molecules (i.e. protein specific antibodies). Cells can be genetically modified to overexpress protein targets, or regulatory components of non-coding sequences, for purposes such as determining fundamental cellular processes, disease mechanisms, gene therapy, and response to therapies. Fusion protein tags can be detected by antibodies or have functional properties to enable localization. Bioluminescent protein and fluorescent protein labeling systems are such genetically engineered optical imaging tools, with greater specificity at lower concentrations than other methods. With this approach, imaging can be in fixed or living cells, tissues or animals. A very attractive feature of the labeling of fusion proteins is that the labeling itself can be restricted to certain locations of a cell (i.e. SNAP-Cell® or SNAP-Surface®). Such discrimination cannot be easily achieved when using bioluminescent proteins. Some fluorescent protein labeling systems enable small nonfluorescent molecules to become fluorescent when bound to a small genetically inserted peptide sequence in the target protein of interest (i.e. FlAsH). Advances in genetically engineered fluorescence systems and microscopy optic machinery, have made imaging a core method for protein localization.

    SNAP-tag®, SNAP-Cell® and SNAP-Surface® are registered trademarks 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.

    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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    FAQs for Protein Localization

    Features of SNAP-tag®/CLIP-tag™

    • 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

    Applications of SNAP-tag®/CLIP-tag™

    • 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

    Advantages of ACP/MCP-tag

    • Small - Expressed tag is only 8 kDa (77aa)
    • Versatile - ACP- and MCP-tagged fusions can be co-expressed and sequentially labeled for two color applications on cell surfaces
    • Specific - Components remain exclusively extracellular, preventing intracellular labeling
    • Precise - Label is covalently bound under biological conditions in a defined position
    • Non-toxic - Substrates are non-toxic to living cells
    • Selection - Choice of substrates available, including 488, 547, 647 nm and biotin

    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.

    Protein Labeling with ACP-tag

    ACP-tag (red) fused to the protein of interest (blue) is labeled in the presence of a required synthase.

    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.