What is the principle of fluorescent dye-labeled antibodies?

Fluorescent labeling is the process of covalently attaching fluorescent groups to molecules such as proteins and nucleic acids. Such processes are typically accomplished using derivatives of the fluorescein group that is capable of selectively reacting with functional groups present on the target molecule.

The most common labeled molecules are antibodies, and labeled antibodies are often used to detect specific target molecules. Fluorescent labels can be used in a variety of detection systems to enable sensitive and quantitative detection and highly specific visualization of cellular components.

Fluorescent dyes for fluorescent labeling are linked to specific proteins or nucleic acids to make them fluorescent. So as long as this substance exists, it will be marked. So another gene created by gene duplication is also marked.

It can be a fluorescent protein such as GFP associated with the protein of interest on the gene. If cloning is not possible, techniques such as immunofluorescent staining are used in tissue samples to visualize the protein of interest. Therefore, antibodies linked to different fluorescent dyes and directly or indirectly bound to the appropriate target structure are used. With the help of fluorescent dyes, fluorescence microscopy is not limited to proteins, but can also be used to detect structures such as nucleic acids and glycans. You can even use a variety of live cell dyes for specific applications, allowing selective staining of organelles (e.g. ER, mitochondria, Golgi apparatus) or functional assays. For example, fluorescence is the readout for live cell tracking, labeling, cell proliferation, or live-dead assays. Even calcium ions and other abiotic matter can be detected.

What is the principle of fluorescent dye-labeled antibodies?

During fluorescence visualization, there are two ways to visualize your protein of interest. Fluorescently labeled antibodies either use an internal fluorescent signal to link the fluorescent protein to the target protein gene or use a fluorescently labeled antibody that binds to the target protein. For some biological problems, post-execution questions are more useful or even necessary. For example, in the case of tissue samples, it is not possible to use fluorescent proteins because the samples are usually derived from organisms that do not contain fluorescent proteins. Another disadvantage of fluorescent proteins is their nature as proteins. With it, they take on specific protein signatures in the cell, which can lead to dysfunction or misinterpretation of the attached protein of interest. However, it should be considered that the use of fluorescent proteins is usually the first choice for studying living cells.

What is the principle of fluorescent dye-labeled antibodies?

Immunofluorescence utilizes the specific binding affinity of antibodies for antigens. This can have two different appearances. The easiest method is to use a fluorescently labeled antibody that binds to the protein of interest. This is called direct immunofluorescence.

In most cases, two forms of antibodies are used. The first binds to the target protein and does not itself have a fluorescent label (an antibody). But the second antibody carries a fluorescent dye specifically to one anti-TB. This method, known as indirect immunofluorescence, has several advantages. On the one hand, there is amplification because there is more than one secondary antibody and one primary anti-TB. On the other hand, secondary antibodies are usually easier to find than specific primary antibodies labeled with common fluorochromes.