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1

Fluorescence spectroscopy relies on?

UV light is absorbed and re-emitted at a longer wavelength (fluorescence

2

in proteins which residue fluoresces

, tryptophan fluoresces

3

Commonly used in which 4 techniques?

3˚ and 4˚ structures
Measuring distances
Catalytic studies
Fluorescence microscopy

4

Excitation excites? and causes what?

Excites the electron in a chromophore into a higher energy state.

5

How do electrons return to ground state after excitation?

Transition vibrations

6

Rigid chromophores tend to have what type of transition vibrations?

Limited range, not possible to return to ground state by vibrations alone.

Instead they undergo: radiative transition (They lose energy by radiation, or light …. A portion of the absorbed energy is re-emitted )

7

Radiative transition?

They lose energy by radiation, or light …. A portion of the absorbed energy is re-emitted

8

Examples of natural fluorescent things?

Fluorescent minerals
Aequoria Victoria (GFP)
Fluorescent fish

9

Biophosphorescence

needs initial light source to activate, but then continues to “glow” after the light source is removed

10

Quinine?

Added to tonic water in order to prevent malaria.
example of a fluorescent compound

11

Quinine absorbs at?

460nm

12

Properties of fluorophores

- chromophore
- delocalised electrons - intense U.V. absorption bands
- ridged
-short excited state

13

Mechanism in which fluorescence is measured?

Quantum yield
where, Q= No. of photons emitted/" absorbed

14

Maximum q value?

1

15

Q is affected by:

Internal factors: distribution of vibrational levels
External factors: quenching

16

Still large q value?

0.1

17

Fluorophores are sensitive to?

environments>>> quenched

18

most useful emission spectra?

Trp q= 0.13

19

fluorescent tags method of detecting proteins ??

The protein of interest is cloned into a vector, so that when it is expressed, it is attached to the fluorescent protein.
When cells are transfected with the DNA (a), when the protein of interest is expressed, so will the fluorescent protein and this can be detected by microscopy.

20

ethidium bromide is ??? how it works

tag ,
intercalates between the bases of DNA, and glows under UV lights – you can use a UV light box to see DNA bands on a gel

21

Ethidum bromide issue? therefore ?

carcinogen, often replaced with SYBR green

22

Fluorescin?

tag

23

Acridine orange works by?

Tag again
intercalates between bases of DNA – used as a cell-cycle marker

24

Spectrofluorimeter works by?

light source
passes through a monochromator (which splits the beam of light into different wavelengths)
through a sample (Light (uv) absorbed then emitted at a longer wavelength which is detected by a photomultiplier)

25

Why are two monochromators required in a Spectrofluorimeter

1. To select wavelength of light required for excitation
2. To select for the wavelength of emitted light

26

At what angle is the emitted radiation detected at?WHY?

The emitted radiation is detected at 90 degrees to the direction of the incident light beam … this is to avoid inadvertent detection of the incident beam.

27

Cuvette most have what property in order to allow what?

the cuvette must have 4 clear sides to allow light through

28

Uses of fluorescent spectroscopy

Structural studies – tertiary and quaternary protein structure
Using tryptophan
FRET: Measuring distances within proteins and complexes
Binding / catalytic studies using a fluorescent substrate
Fluorescence microscopy

29

What qualities within the quaternary structure of proteins can be detected using Structural fluorescence spectroscopy

Folding / conformational state / monomer association / ligand binding

30

changes in structure can cause what to happen to tryptophan fluorescence?

changes in intrinsic tryptophan fluorescence (used as a reporter group)