Question

4. What is the difference between a fluorescent-tagged protein, fluorescent-tagged antibody, and a fluorescent dye? What is one advantage and one disadvantage of each? (6 marks)

Answer 1

Answer :- Fluorescent colorants offer greater photostability and brightness compared to fluorescent proteins and do not need a maturation period. Nonetheless, antibody conjugates or peptide tags usually target fluorescent colorants to proteins of interest.

A fluorescent tag, also called as luorescent label or fluorescent sample, is a molecule in molecular biology and biotechnology which is chemically bound to help identify a biomolecule such as an antigen, an antibody, or amino acid. GFP's best advantage is that it can be heritable, depending on how it was inserted, allowing continued analysis of the cells and tissues in which it is expressed. GFP visualization is non-invasive and requires only blue-light illumination. disadvantages of GFP fusion proteins are that they are usually over-expressed compared to endogenous proteins, and the GFP tag will, in theory, affect protein functioning. They are also poisonous, and interfere with normal biological processes at times.

Fluorescent tagging or labeling typically utilizes a reactive version of a fluorescent molecule known as a fluorophore. The most widely branded molecules are antibodies, proteins, amino acids, and peptides that are then used to identify a specific target. On a microscope slide, the fluorescent antibodies bind to the bacteria, allowing for ready identification of the bacteria using a fluorescence microscope. Therefore, the DFA technique is useful for the visualization of certain bacteria which are difficult to isolate from patient samples or culture. There are some drawbacks in this approach though. Because the number of fluorescent molecules that can be connected to the primary antibody is small, direct immunofluorescence is significantly less sensitive than indirect immunofluorescence and may lead to false negatives.

Fluorescent dyes are non-protein molecules which absorb light at a longer wavelength and re-emit it. These are often used in biomolecular fluorescent labelling and may be smaller or more photostable than but not genetically encodable fluorescent proteins. Fluorescent dyes have many advantages as one of the commonly used fluorescent labeling compounds (fluorescence dye probe), such as fast detection speed, good repeatability, low dosage, and non-radiation.