Fluorescence and Immunofluorescence Techniques

Flashcards covering fluorescence, immunofluorescence techniques, and FRAP for studying membrane fluidity.

What is a fluorophore?

A fluorescent dye molecule that absorbs light at an excitation wavelength and emits light at an emission wavelength.

What are the two main conditions for successfully combining multiple fluorophores in imaging?

Each selected fluorophore must emit at a different wavelength with minimal overlap, and there must be minimal overlap of excitation wavelength with emission wavelength of a different fluorophore.

What is immunofluorescence?

A technique that leverages antibody-antigen interactions to conjugate a fluorophore to a target protein.

How does direct immunofluorescence work?

A fluorescently-conjugated primary antibody binds directly to the antigen, attaching the fluorophore to the antigen.

How does indirect immunofluorescence work?

A primary antibody binds to the antigen, and then a fluorescently-conjugated secondary antibody recognizes the host species of the primary antibody, attaching the fluorophore.

What are the advantages of direct immunofluorescence?

It is faster, easier, and has less likelihood of non-specific (background) signal.

What is an advantage of indirect immunofluorescence over direct immunofluorescence?

It is more sensitive and produces a stronger signal due to the amplification step provided by the secondary antibody.

On what types of cells can direct and indirect immunofluorescence be performed?

Live cells (if targeting a cell surface expressed antigen) or fixed and permeabilized cells (if targeting an intracellular antigen).

What technique did Frye and Edidin use to force two cells to fuse together in their 1970 study of plasma membrane structure?

Zellfusion, using Sendai virus.

What modern method is used for quantifying the fluidity of the plasma membrane?

Fluorescence Recovery After Bleaching (FRAP).

In a FRAP experiment, what does a higher Diffusion Coefficient indicate?

Increased energy of movement of labeled molecules and increased membrane fluidity.

In a FRAP experiment, what does a higher Mobile Fraction indicate?

An increased number of labeled molecules available to move and increased membrane fluidity.

How might a drug that increases expression of an unsaturated phospholipid affect diffusion coefficient and/or mobile fraction in a FRAP experiment?

It would likely increase both the diffusion coefficient and the mobile fraction, as unsaturated phospholipids generally increase membrane fluidity.