Fluorescence Flashcards
What do macromolecules absorb?
Electromagnetic radiation
How many molecules can spectroscopy measure?
Multiple
What’s one way to measure reaction kinetics?
Coupling to a known chromophore
What is the Beer-Lambert relationship?
Absorbance(lambda) = epsilon(lambda) x concentration x path length
What wavelength do peptide bonds absorb?
214nm
What wavelength do aromatic amino acids absorb?
280nm
What is protein absorption affected by?
The microenvironment
What wavelength do nucleic acids absorb?
~260nm
What does absorbance do as DNA is heated?
Absorbance increases as temperature increases due to the change from double stranded to single stranded
What is Tm?
Melting temperature
The value at which 50% has denatured
Give a use of Tm
The ratio of A-T and G-C base pairs
What is the formula for energy absorbed?
Eabsorbed = heat x concentration/wavelength
What is the formula for energy emitted?
hc/lambda
How is energy lost when the excited electron moves back to the ground state?
As heat and as fluorescence
Describe the relationship between energy and wavelength
Lower energy = longer wavelength
What spectra do fluorescent molecules have?
Absorption and emission
What is the Stokes shift?
The difference between the wavelengths of absorption and emission
What is more sensitive? Fluorescence or absorbance
Fluorescence
What is the set up of a basic fluorometer?
Light source
Monochromator
Sample
Sensor
Name some uses of fluorescence
Cellular localisation Biosensor Gene expression Membrane potentials Cell separation and counting Ca2+ imaging
What is GFP?
Green fluorescent protein
Emission at 509nm
