Biochemical methods

Question 1

What is UV/vis spec used for in biosciences?

Answer 1

Following enzyme activity or looking at how much substance there is.

Question 2

What happens when UV is absorbed?

Answer 2

Electrons are promoted to a higher energy.

Question 3

What is the apparatus required for UV /Vis spec?

Answer 3

light source–> entrance slit–> monochromator (splits light)–> exit slit–> sample–>detector.

Question 4

What light sources can be used for UV?

Answer 4

Tungsten bulb: 400-700nm
Deuterium lamp: 200-400nm

Question 5

What cuvettes are required?

Answer 5

Quartz/ special plastic for UV

Question 6

What 2 measurements are made by a UV spec?

Answer 6

Absorbance: How much light is absorbed, no units
Transmittance: How much light goes through %

Question 7

What is the equation for absorbance?

Answer 7

A=ecl
A= absorbance
e= extinction coeff
c= concentration
l= path length

Question 8

What is absorbance in terms of transmittance?

Answer 8

A= log10(I0/I)

Question 9

What wavelength does DNA absorb at?

Answer 9

bases absorb at 260nm

Question 10

What wavelength does a peptide bond absorb at?

Answer 10

peptide bond: 190nm

Question 11

What wavelength do Tryptophan and Tyrosine absorb at?

Answer 11

280/274 nm

Question 12

What wavelength does prosthetic groups Haem and NADH absorb at?

Answer 12

Haem: 400nm
NADH=340nm

Question 13

What is an issue with UV/Vis spec and contamination?

Answer 13

Light scattering due to pptx and dust, bubbles, degrade, flouresence.

Question 14

How does Flourescence work?

Answer 14

Where a sample absorbs a wavelength, electrons get excited and lose energy, electrons lose wavelength and go back down to their original state, light is emitted when they change but this is of lower energy and higher wavelength.

Question 15

What are some natural flourescents?

Answer 15

GFP, aspirin, Vitamin A

Question 16

What are some features of flourescent molecules?

Answer 16

1. Alternating system with alternating single + double bonds
2. Has aromatic rings
3. Has electron donating and electron attracting groups
4. Has a rigid planar structure

Question 17

Why is flourescence used?

Answer 17

It is used to detect molecules in a sample, but it is at a higher sensitivity and increased specificity because 2 wavelengths are measured. However, it can easily be disturbed.

Question 18

What is stokes shift?

Answer 18

When the molecules are put through flourescence, two values are taken. One is the value agt which it is when excited, and the other when it drops. Two peaks are produced, one at a higher wavelength than the other. The higher the difference between the peaks, the higher the stokes shift and the more useful the flourescence. / more sensitive the assay.

Question 19

How does Size exclusion chromatography work?

Answer 19

Tiny beads that are the matrix (stationary phase), they have tiny pores in them that allow for the smallest molecules to go through.

Question 20

In size exclusion chromatography, which molecules are eluted first?

Answer 20

The largest molecules are eluted first because they cannot fit through the pores in the beads so join the stationary phase and are eluted.

Question 21

What is void volume?

Answer 21

It is the largest molecule that comes straight through.

Question 22

What can size exclusion chromatography be used for?

Answer 22

Calculation of molecular mass: where known masses can be eluted and volume of each measured. Then plot graph of Volume eluted vs log of molecular mass and find the unknown mass.
Oligomerisation: you can figuire out whats in an oligomer etc

Question 23

Advantages and disadvantages of size exclusion chromatography?

Answer 23

Cheap and easy
slow, not good resolution

Question 24

How do you improve resolution in size exclusion chromatograhpy?

Answer 24

FPLC- Fast protein liquid chromatography
HPLC- High pressure liquid chromatography
Use bigger columns.

Question 25

How does electrophoresis work?

Answer 25

Agarose or polyacrylamide gels are used which have a mesh like structure and cause proteins to move through them. Smaller proteins move faster and larger proteins move slower. The concentration of agarose/gel can be changed and a gradient gel can also be used.

Question 26

What is the process of gel electrophoresis?

Answer 26

Add agarose gel, comb to cause wells. add dna with loading dye (glycerol and bromophenol blue). Also add DNA with known size. Then add ethmidium bromide which is a flourescent dye, use UV light to visualise it.

Question 27

How is SDS page used?

Answer 27

It is used when the proteins have different charge to mass ratios or shapes etc. SDS (sodium dodecyl sulphate) is used to denature all proteins and add many negative charges to them. The glycerol is added for density, Bromophenol blue is added for visualisation, Beta-me/DTT used to break disulphide bonds. The solution is also heated to ensure denaturation of proteins.

Question 28

How do we visualise the proteins in SDS-Page

Answer 28

Stain such as coomassie blue is used, destain is used to remove all the extra stain.

Question 29

How does Ion exchange work?

Answer 29

Beads with a group attached on it that has a charge is filled into a tube.

Question 30

What charge beads are filled into ion exchange for anion exchange resin?

Answer 30

Positively charge beads so anions can stick and the positive ions can be eluted.

Question 31

What charge beads are filled into Ion exchnage for cation exchange resin

Answer 31

Has negatively charged beads so positive proteins stick and the other eluted out.

Question 32

What is the isoelectric pH

Answer 32

The pH at which the overall charge on the compound is neutral.

Question 33

What charge does the does a molecule when above the PI?

Answer 33

Negative

Question 34

What charge does the does a molecule when below the PI?

Answer 34

Positive

Question 35

What does the pH have to be compared to PI for anion exchange resin and cation exchange resin?

Answer 35

Anion: pH>PI Cation: pH

Question 36

Isoelectric focussing- how does it work?

Answer 36

Silica support covered in hydrophobic phase making the entire bead hydrophobic. This means polar molecules elute first and hydrophobic molecules take longer. pH=pI=0 net charge, meaning there is no electrophoretic velocity.

Question 37

How does affinity chromatography work?

Answer 37

Where there are beads and ligands bound to it, the ligand sticks to certain proteins. When an excess of those ligands are added , they compete for a space and the proteins fall off.

Question 38

How do Western Blots work?

Answer 38

It is a way to visualise a specific protein in an SDS page. However after getting the SDS page, don't stain it. Stack the gel with PVDF membrane. Wash membrane with 4% milk or BSA to block all protein sites. Add protein specific antibody (1) . Then wash so all non specifically bound antibody is washed off. Then add in (2) enzyme linked antibody which recognises (1). Then it is washed again and place in substrate that reacts wit enzyme. Enzyme 2 should be an antibody from another animal.

Question 39

How does ELISA work?

Answer 39

Indirect: antigen coats bottom, block with non specific protein, wash, add enzyme linked 2 antibody, Changes colour when enzyme bound due to substrate and measures absorbance. Sandwich: Same thing but block with the antigen and the enzyme should bind to antigen.