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Biomolecules > Biochemical methods > Flashcards

Biochemical methods Flashcards

1
Q

What is UV/vis spec used for in biosciences?

A

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

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2
Q

What happens when UV is absorbed?

A

Electrons are promoted to a higher energy.

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3
Q

What is the apparatus required for UV /Vis spec?

A

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

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4
Q

What light sources can be used for UV?

A

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

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5
Q

What cuvettes are required?

A

Quartz/ special plastic for UV

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6
Q

What 2 measurements are made by a UV spec?

A

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

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7
Q

What is the equation for absorbance?

A

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

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8
Q

What is absorbance in terms of transmittance?

A

A= log10(I0/I)

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9
Q

What wavelength does DNA absorb at?

A

bases absorb at 260nm

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10
Q

What wavelength does a peptide bond absorb at?

A

peptide bond: 190nm

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11
Q

What wavelength do Tryptophan and Tyrosine absorb at?

A

280/274 nm

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12
Q

What wavelength does prosthetic groups Haem and NADH absorb at?

A

Haem: 400nm
NADH=340nm

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13
Q

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

A

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

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14
Q

How does Flourescence work?

A

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.

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15
Q

What are some natural flourescents?

A

GFP, aspirin, Vitamin A

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16
Q

What are some features of flourescent molecules?

A
  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
17
Q

Why is flourescence used?

A

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.

18
Q

What is stokes shift?

A

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.

19
Q

How does Size exclusion chromatography work?

A

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

20
Q

In size exclusion chromatography, which molecules are eluted first?

A

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

21
Q

What is void volume?

A

It is the largest molecule that comes straight through.

22
Q

What can size exclusion chromatography be used for?

A

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

23
Q

Advantages and disadvantages of size exclusion chromatography?

A

Cheap and easy
slow, not good resolution

24
Q

How do you improve resolution in size exclusion chromatograhpy?

A

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

25
Q

How does electrophoresis work?

A

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.

26
Q

What is the process of gel electrophoresis?

A

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.

27
Q

How is SDS page used?

A

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.

28
Q

How do we visualise the proteins in SDS-Page

A

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

29
Q

How does Ion exchange work?

A

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

30
Q

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

A

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

31
Q

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

A

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

32
Q

What is the isoelectric pH

A

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

33
Q

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

A

Negative

34
Q

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

A

Positive

35
Q

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

A

Anion: pH>PI
Cation: pH<PI

36
Q

Isoelectric focussing- how does it work?

A

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.

37
Q

How does affinity chromatography work?

A

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.

38
Q

How do Western Blots work?

A

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.

39
Q

How does ELISA work?

A

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.

Biomolecules (3 decks)

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