Cellular Analysis
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  • Pulse Chase

    Pulse-chase experiments use labeled compounds to follow the dynamics of cellular processes and pathways. Molecules in a cell are continually being synthesized and degraded at various rates. Changes in molecule localizations and expression levels over time, can be detected by first “pulsing” or exposing cells to a labeled compound, then sequentially exposing the cells to the same compound, unlabeled, which is the “chase”. Compounds are commonly labeled with radioisotopes or fluorescent dyes. Turnover can be detected visually by microscopy techniques.

    Various protein labeling methods for pulse-chase have specific application potentials. Reporter fluorescence fusion proteins are often used, but they can disrupt the native target protein's complexing behavior, be toxic to the cells, or affect target stability for pulse chase experiments. Fluorescence reporter fusion proteins also cannot be quenched. SNAP-tag® and CLIP-tag™ are useful for multicolor pulse-chase experiments. Sequential labeling with two or more fluorophores is possible. The non-overlapping substrate specificity of SNAP-tag and CLIP-tag permit simultaneous pulse-chase experiments to visualize different generations of two different proteins in one cell, further increasing the potential of the approach (1). Unlike with reporter fluorescence fusion proteins, like GFP, labeling of newly synthesized proteins can also be turned off using available blocking reagents (e.g., SNAP-Cell® Block). For development of pulse-chase techniques in thick tissues or animals (2), TIME-stamp tagging has advantages. Scalable proteomic analysis, detected by mass spectrometry, has been developed using a azidohomoalanine (azhal) label for rapid pulse-chase (3).

    SNAP-tag® and SNAP-Cell® are registered trademarks of New England Biolabs, Inc.

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

    References

    1. Gautier, A., Juillerat, A., Heinis, C. et al. (2008) Chem. Biol. 15, 128. PMID: 18291317
    2. Lin, M.Z. And Tsien, R.Y. (2010) Curr Protoc Protein Sci. Chapter 26, Unit 26.5. PMID: 20155731
    3. Kramer, G. Kasper, P.T., de Jong L, de Koster CG (2011) Methods Mol Biol. 753, 169-81. PMID: 21604123

    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 Pulse Chase

    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.