Cellular Analysis
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  • Receptor Internalization

    Membrane receptor internalization studies follow the functional process of receptors binding to ligands or agonists. Receptor internalization, or trafficking, is a part of cell signaling. Cancer, psychoactive drug targets, virology, endocytosis, neurotransmitters and addiction are all relevant areas of study. However, membrane receptors are challenging to study given their low native expression levels.

    Recombinant bioluminescent reporters and fluorescent protein labeling systems have revolutionized the study of receptor internalization. These systems can be used instead of traditional, cumbersome radioactively tagged ligand probes. Fluorescent probes are localized in cells through a process called “gating”, to be evaluated by ELISA, flow cytometry, imaging or high-throughput assays (1). Real time receptor internalization methods using time lapse confocal microscopy require the use of recombinant bioluminescent reporter proteins, protein labeling systems or antibodies. Bioluminescent reporters fluoresce regardless of receptor localization. Therefore, applications like flow cytometry or plate readers will not discriminate sub-cellular or membrane localization. To overcome this issue, SNAP-tag®, CLIP-tag™, ACP-tag and MCP-tag protein labeling systems, as well as fluorogen activating proteins (FAPs) (2), have membrane impermeant substrates. Simultaneous localization of more than one protein is possible with these substrates when targets are engineered into different systems with unique substrate requirements. These systems also have the advantage of being able turn on the signal at will, allowing time-resolved analysis of receptor internalization and protein trafficking which can be combined with FRET analysis (2).

    References

    1. Evans, N. (2004) Current Protocols in Pharmacology Unit 12.6. PMID: 22294118
    2. Saunders, M.J. (2012) Methods, 2012 Feb 16. PMID: 22366230
    3. Comps-Agrar L. et al (2011) Methods Mol Biol. 756, 201-214. PMID: 21870227

    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.

    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 Receptor Internalization

    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.