Question

Please explain how a fluorescent tag can be used to visualize protein localization in a cell.

Answer 1

Solution

• In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore.
• The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.
• Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
• GFP coding sequences have been fused at either the 5′ or 3′ end of the coding region of a DNA sequence of interest, leading to production of N‐terminal or C‐terminal fusions of GFP. Such chimeric genes have been incorporated into stable transgenic plants, or introduced into plant cells for transient assays.
• Production of stable transgenic plants has the advantage that many different cell types can be examined in which the GFP/protein fusion is expressed, while not all cell types are technically suitable for transient expression
• Chemical tags have been tailored for imaging technologies more so than fluorescent proteins because chemical tags can localize photosensitizers closer to the target proteins.
• Proteins can then be labeled and detected with imaging such as super-resolution microscopy, Ca2+-imaging, pH sensing, hydrogen peroxide detection, chromophore assisted light inactivation, and multi-photon light microscopy. In vivo imaging studies in live animals have been performed for the first time with the use of a monomeric protein derived from the bacterial haloalkane dehalogenase known as the Halo-tag.
• The Halo-tag covalently links to its ligand and allows for better expression of soluble proteins.