Analytical Techniques In Biochemistry-Fluorescence

Front 1

What 2 states can molecules exist in?

Back 1

Ground state and excited state

Front 2

S0 denotes what?

Back 2

Lowest vibrational level of the ground state

Front 3

In order to pass to excited level, what needs to happen to the molecule?

Back 3

Absorbtion of a photon at a specific wavelength

Front 4

Absorbtion of a photon takes how long?

What happens next?

How long does this take?

Back 4

10^-15 seconds

Will revert to minimum vibrational energy level for excited state

10^-12 seconds

Front 5

Following being at lowest vibrational energy level for the excited state, what happens next?

Back 5

Will revert to any vibrational level of ground state, emitting a photon (of lower energy and higher wavelength) than one absorbed

Front 6

What follows the drop to any vibrational level of the ground state?

Back 6

Molecule relaxes to lowest vibrational level of ground state

Front 7

Not all molecules loose their energy through fluorescence. What happens to these molecules? How do they lose their energy?

Back 7

Lose energy by non-radiative relaxation, emit through thermal energy rather than light

Front 8

Define 'Fluorescence Intensity'.

Back 8

The amount of energy emitted by a fluorophore

Front 9

Define 'Quantum Yield'.

Back 9

Ratio of emitted light to absorbed light

Front 10

State 2 conditions where fluorescence can be quenched. What can be done to both to prevent this?

Back 10

1. Collisions with other molecules: need to keep temperature low

2. Chemical reactions with added molecules: keep sample neutral or slightly basic

Front 11

Sensitivity of fluorescence, in what range?

Back 11

Nanomolar

Front 12

Fluorescent molecules often have conjugated bonds with associated pi orbitals. What does cpnjugated mean?

Back 12

Alternating double and singe C-C bond

Front 13

Why does a planar ring increase fluoresence?

Back 13

Annular cloud above and below the plane of the ring

Front 14

Fluorescein produces green light from blue light. What is ethidium bromide used for?

Name 3 other fluorescent molecules.

Back 14

DNA staining

Tryptophan, BODIPY and Dansyl

Front 15

In the instrumentation of fluorescence, what is the monochromator used for?

Back 15

Blocks all light above and below the target frequency

Front 16

What conditions must the sample be kept in?

What is the purpose of the emission monochromator?

Back 16

Dilute and cold

Blocks light of frequency from 1st monochromator

Front 17

The emission data provides a larger spread than absorbtion data, why is this?

Back 17

Large variety of vibrational levels it can land on at the ground state

Front 18

State 3 TYPES of biomolecules that are naturally fluorescent. Give 2 examples of each.

Back 18

Answers can include any of the following:

Aromatic amino acids-Phenylalanine, Tryptophan and Tyrosine

Purine/Pyrimidine bases in DNA-Adenine, Guanine, Cytosine and Uracil

Some coenzymes-FAD, NAD and Flavins

Front 19

Many fluorescent dyes have enhanced fluoresence when they are _ or _. What 2 conditions can increase fluorescence?

Back 19

Non-polar solution or bound in a rigin hydrophobic environment

Front 20

In molecular beacons, to break apart the complementary binding to itself, what needs to happen?

Back 20

Increase tmperature to break Hydrogen bonds

Front 21

How is light emission possible with molecular beacons?

Back 21

Hybridisation and seperation of quencher and fluorophore

Front 22

State 2 biological sequences that can be detected using molecular beacons?

Back 22

Cystic fibrosis and anthrax

Front 23

Molecular beacons are used in genomics and pharmacogenetics for the detection of specific sequences. What else can they detect? (2 things)

Back 23

Single nucleotide polymorphisms

Real time monitoring of PCR

Front 24

Binding of ethidium bromide to DNA causes?

How is this caused?

What is seen as a result?

Back 24

Partial unwinding of the double helix

Intercalation between adjacent base pairs

Fluorescence increases

Front 25

Where is EtBr fluorescence used in the labratory? (2 things)

Back 25

Detection of DNA in gels and monitor processes where DNA strands hybridise or are seperated

Front 26

A DNA strand to be sequenced is copied in the presence of a small amount of what?

What is this purpose of this^?

Back 26

Fluorescent labelled dideoxynucleotide triphosphates

Stop replication at 3' terminus

Front 27

How does this help in sequencing?

Back 27

Different fluorescents are used for Adenine, Uracil, Cytonsine and Guanine

Read off colours as they come off

Front 28

Using a computer, how many base pairs can be obtained in one run, instead of standard gel electrophoresis?

Back 28

750 base pairs rather than 250

Front 29

Fluorescence microscopy has 3 main stages. For the fluorophore 'fluorescein', stage one includes a barrier that allows light with a wavelength above 450nm, but below 490nm. What occurs at step 2?

Back 29

Beam splitting mirror reflects down wavelength less than 510nm onto sample, but allows wavelength greater than 510nm to pass through upon reflection from sample

Front 30

What is the 3rd and final step of fluorescence microscopy?

Back 30

Second barrier cuts out unwanted signals, allows wavelength greater than 520nm, but less than 560nm to pass through

Front 31

An amino acid can be converted into a fluorophore using just O2, what 3 processes cause this?

What chemical structure is made?

Back 31

Cyclisation, dehydration and oxidation

5 membered ring

Front 32

Which 3 amino acids can be converted?

Back 32

Tyrosine, glycine and thiamine

Front 33

20% of a population of chromophores will excite at a higher wavelength of light. What has this different population undergone?

Back 33

Deprotonation

Front 34

Native GFP has what kind of structure?

In solution, in what form does it exist?

What is its fluorescence sensitive to?

Back 34

Beta barrel structure

Exists as a dimer in solution

Cl- and other anions

Front 35

What is the following describing:

'technique in which energy emitted from one fluorophore causes the excitation of a second, longer wavelength fluorophore'?

Back 35

Fluorescence resonance energy transfer

Front 36

What 3 conditions are needed for FRET to occur?

Back 36

1. 2 proteins should have different fluorophores attached

2. Emission spectrum of donor must overlap with excitation of the acceptor

3. 2 proteins must come within 1-10nm of each other

Front 37

What 2 things can FRET be used for?

Back 37

1. Determine distance between donor and acceptor

2. Quantify binding of a fluorescent ligand to a fluorescent receptor

Front 38

Fura-2 will produce fluorescence upon binding of which ion?

Back 38

Ca2+

Front 39

Fura-2 used in combination with GFP makes it possible to moniter 2 things, what are they?

Back 39

Simultaneous receptor localisation and cell activity

Front 40

What does FRAP stand for?

What is it used to study?

Back 40

Fluorescence Recovery After Photobleaching

Localisation and kinetic behaviour of FP-tagged proteins

Front 41

Quantitative studies can provide an estimation of what 2 things for a protein?

Back 41

Effective diffusion co-efficient

Mobile fraction

Front 42

Define percentage recovery in an equation?

Back 42

Fluorescence returned/fluorescence lost x100

Front 43

What does FLIM stand for?

What is it used to measure?

Back 43

Flourescence Lifetime Imaging Microscopy

The time taken for a fluorophore to become excited and return to the ground state

Front 44

What is the main difference between time domain fLIM and frequency domain FLIM?

Back 44

Time: lifetime measured in nanoseconds

Frequency: Lifetime calculated by measuring the phase shift of fluorescence and the reduction in amplitude

Front 45

RNH2 can produce a fluorescent product from which substrate?

Back 45

Fluorescein isothiocyanate

Front 46

RSH can produce a fluorescent product from which substrate?

Back 46

Fluorescein iodoacetamide

Front 47

Protein folding can be monitored by an increase in what?

What conditions are needed for this to happen?

Back 47

Tryptophan fluorescence

If the tryptophan residue is buried in the hydrophobic core during folding

Front 48

If tryptophan is near the ligand binding site, then ligand binding can be monitored using what?

Back 48

Ligand induced quenching or enhancement of fluorescence