What is Fluorescein Isothiocyanate (FITC)?
Fluorescein Isothiocyanate (FITC), a derivative of fluorescein, this fluorescent cyanine dye binds to amino groups in antibodies, making it an integral player in immunofluorescence techniques. Understanding FITC and how it functions in various applications is vital to the ongoing advancement of protein research, pharmaceutical development, and medical diagnostics.
Between Fluorescein and FITC: Notable Differences
Though FITC is derived from fluorescein, there are crucial differences that set these two compounds apart. Where fluorescein is a xanthene dye showcasing broad spectrum fluorescence emission, FITC introduces an isothiocyanate group, enabling the molecule to act as a hapten. Unlike fluorescein, FITC can be directly conjugated to antibodies, securing its place in biochemistry as a valuable fluorescent labeling agent.
How does FITC Labelling Work?
FITC Labelling is a technique used in immunology, biochemistry and cell biology to attach a fluorescent dye to a biological target such as a protein or antibody. The purpose is to enable the detection and measurement of this target using a technique known as fluorescence microscopy or flow cytometry.
The mechanism of FITC labelling involves a chemical reaction between the isothiocyanate group on the FITC molecule and an amino group, typically the side chain of a lysine residue, on the protein or antibody.
Here's the general procedure:
1. Dissolve the protein or antibody in a mild bicarbonate buffer. This will ensure that the proteins are in their native state and the amino groups are available for labelling.
2. Add the FITC solution to the protein solution while stirring.
3. The FITC molecules will start to react with the protein molecules. This reaction will form a thiourea bond between the FITC and protein, effectively labelling the protein.
4. The reaction is allowed to proceed for a several hours.
5. After the reaction is complete, the labelled proteins are separated from the unreacted FITC molecules using dialysis or gel filtration.
Once labelled, the FITC-protein conjugates can be visualized under a fluorescence microscope, where they will emit green light upon excitation with blue light. This allows scientists to track the location and movement of these proteins within a cell or tissue sample. FITC labelling also allows for quantitative measurements of protein concentrations in a sample, as the intensity of the fluorescence is directly proportional to the amount of protein present.
What is FITC Used for?
Applications of FITC are multifaceted due to its efficient fluorescent properties. In biochemistry, the molecular structure of FITC allows it to be used for staining cells, creating a fluorescence that can be detected and analyzed. This has profound implications inareas such as histology and cell biology.
Adding to this, another key application of FITC is in Flow Cytometry – a technique used to measure physical and chemical characteristics of a population of cells or particles. FITC's ability to bind to antibodies renders it instrumental in identifying and sorting different cell types in a sample.
Furthermore, FITC is used for detecting microorganisms in heterogeneous samples. Again, its use as a fluorescent marker, coupled with its ability to bind to antibodies, simplifies the process of identifying and quantifying microbes in given samples.
Reference
- Gu, R.; et al. A synthesized glucocorticoid-induced leucine zipper peptide inhibits retinal Müller cell gliosis. Frontiers in Pharmacology 9 (2018): 331.
