Use of Ethidium Bromide in gel electrophoresis Ethidium bromide is an

intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis.

When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA.

Ethidium bromide is commonly used to detect nucleic acids in molecular biology laboratories.

In the case of DNA this is usually double-stranded DNA from PCRs, restriction digests, etc.

Detection typically involves a gel containing nucleic acids placed on or under a UV lamp.

Gels stained with ethidium bromide are usually viewed indirectly using an enclosed camera, with the fluorescent images recorded as photographs.

DNA is stained using ethidium bromide

How do ethidium bromide fluoresce? The reason for ethidium bromide's intense

fluorescence after binding with DNA appears to be the hydrophobic environment found between the base pairs.

By moving into this hydrophobic environment and away from the solvent, the ethidium cation is forced to shed any water molecules that were associated with it. As water is a highly efficient fluorescent quencher, the removal of these water molecules allows the ethidium to fluoresce.

Quenching A quencher is a substance that absorbs

excitation energy from a fluorophore and dissipates the energy as heat; or re-emits much of this energy as light .

Quenchers are used in molecular biology in conjunction with fluorophores. When the two are close together, such as in a molecule or protein, the fluorophore's emission is suppressed. This effect can be used to study molecular geometry and motion

Quenching

Quenching can be caused by chemical reactions of fluorescent species with added molecules, transfer of energy to other molecules by collision and transfer over distance.

The reverse of quenching;enhancement of fluorescent intensity is observed in some situations.

9Dr. Nikhat Siddiqi

Several fluorescent dye molecules are quenched in aqueous solution, but their fluorescence is greatly enhanced in a non polar or rigidly bound environment eg interior of protein.

Both quenching and fluorescence enhancement can yield information about the structure of the molecule.

10Dr. Nikhat Siddiqi

Immunofluorescent Staining• Immunofluorescent staining makes use of antibodies to locate and identify patterns of protein expression in cells.

•Primary antibody binds to antigen.

•Antibody-antigen complex is bound by a secondary antibody •conjugated to a fluorochrome.

Upon absorption of high energy light, the fluorochrome emits light at its own characteristic wavelength (fluorescence) and thus allows detection of antigen-antibody complexes.

Basic Staining Technique

Immunofluorescent staining of nerve cells