In the dynamic fields of molecular biology and cell biology, precision in labeling and tracking proteins stands as a cornerstone. This capability serves as the linchpin for researchers, offering insight into the intricate functions, interactions, and cellular whereabouts of these pivotal biomolecules. One of the most powerful techniques for protein labeling involves the use of near-infrared (NIR) fluorescent probes. These probes offer several advantages, including minimal cellular autofluorescence and the ability to penetrate deeper tissues. In this article, we will explore how different protein labeling systems, such as SNAP-tag, CLIP-tag, Halo-tag, PYP-tag, and BL-tag, utilize various NIR fluorescent probes to shed light on the world of protein research.
Fig 1. Schematic representation of self-labeling protein tag with chemical probe. (Reja SI et al. 2020)
SNAP-tag and CLIP-tag Based Systems
SNAP-tag and CLIP-tag are versatile protein labeling systems that utilize a variety of NIR fluorescent probes to label target proteins. The SNAP-tag system hinges on the SNAP protein, forming covalent bonds with O6-benzylguanine (BG) derivativesn. Similarly, the CLIP-tag system relies on the CLIP protein, engaging in its own covalent association, this time with O2-benzylcytosine (BC) derivatives. These two systems, remarkable in their design, find applications spanning live-cell imaging and in-depth protein trafficking analysis.
NIR fluorescent probes compatible with SNAP-tag and CLIP-tag systems include BG- and BC-conjugated dyes such as BG-647 and BC-800. Emitting within the near-infrared spectrum, these probes illuminate the path to deep tissue imaging while minimizing background disturbances. What amplifies their utility is the versatility to choose from a diverse repertoire of NIR probes, rendering them indispensable tools in the realm of protein labeling and tracking.
Halo-tag-based System
The Halo-tag system is another powerful tool for protein labeling, offering versatility and specificity. Central to this system is the HaloTag protein, a molecular entity known for its remarkable ability to establish covalent bonds with its counterpart, the HaloTag ligand. This nuanced interaction forms the cornerstone of precise and high-affinity protein labeling when coupled with NIR fluorescent probes.
PYP-tag-based System
The PYP-tag system, though somewhat obscure in the realm of protein labeling, offers substantial value to the scientific community. It hinges on the Photoactive Yellow Protein (PYP), a petite yet highly specialized protein with a remarkable affinity for its target ligand. Through meticulous engineering, researchers harness the PYP protein's unique attributes to precisely label proteins of interest.
Notably, the PYP-tag system accommodates NIR fluorescent probes, such as PYP-655 and PYP-750, emitting in the near-infrared spectrum. These probes bring the advantage of exceptional photostability and minimal photobleaching, rendering them ideal for prolonged live-cell imaging investigations. The distinctive characteristics of the PYP-tag system position it as a promising tool for probing protein dynamics and cellular localization.
BL-tag-based System
The BL-tag system is a robust protein labeling technique that relies on the biotin ligase enzyme. In this system, a small peptide sequence called the BL-tag is fused to the protein of interest. When biotin is supplied, the biotin ligase enzyme specifically attaches biotin molecules to the BL-tag. These biotin-labeled proteins can then be detected using fluorescently labeled streptavidin or avidin.
The advantage of this system lies in its simplicity and high specificity. Researchers can label proteins of interest with biotin and then detect them using the appropriate NIR-labeled streptavidin or avidin conjugates. This system is widely used in various applications, including Western blotting and immunofluorescence assays.
Reference
- Reja SI, Minoshima M, Hori Y, Kikuchi K. Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications. Chem Sci. 2020; 12(10):3437-3447.
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