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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® and CLIP-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.

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FAQs for Receptor Internalization
Protocols for Receptor Internalization
Application Notes 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
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.
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Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.


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