Fluorescence Flashcards
What is the substance known as that you can couple to a reaction so that reaction kinetics can be measured?
chromophore
What is the slope equal to on an absorbance/time graph?
absorbance/min
Does single stranded DNA have higher or lower absorbance?
higher
What is the melting temperature (Tm)?
the temperature at which 50% is denatured
What can you use the thermal denaturation of DNA to investigate?
- work out the GC/AT ratio because GC have three H bonds holding them together
- if Tm is higher likely more GC
What are intercalators?
compounds that stick DNA together and stop them from turning into single stranded DNA during replication (cancer treatment)
What is an emission spectra?
Excited electrons that have absorbed energy can return to their ground state.
Why is excitation sometimes not equal to emission?
because some energy can be lost as heat
What is the stoke shift?
the difference between the wavelength of the absorption and emission spectrum
Why is fluorescence more sensitive than absorption?
it is a cyclical process
What is the emission intensity related to?
excitation intensity
How do you measure fluorescence?
With a Fluormeter
In a fluorometer, why is the sensor at right angles to the sample?
increases signal to noise ratio (fluorescence travels in all angles)
What is the difference between intrinsic and extrinsic fluorophores?
- intrinsic when the absorbance is being measured by something within a compound e.g an aromatic ring in a protein
- extrinsic is when a compound has to be tagged with something that will show absorbance
What are the uses of fluorescence?
- protein localisation and conformation (different amino acids will have different emissions)
- ligand binding
- mutations
- drug screening
- cellular localisation tag with green fluorescent protein (GFP)
- cell separating and counting using fluorescence activated cell sorting machines
- Ca2+ chelators-visualising intracellular Ca2+ levels (work out free Ca2+ levels in cytoplasm)
