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BB10186 > Lec 2 Crennell > Flashcards

Lec 2 Crennell Flashcards

1
Q

What is involved in the clarification of proteins?

A
  • removing cell debris and other particulates
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2
Q

Once you have got rid of particulates and aggregates what must you not do to get rid of nucleic acids?

A
  • don’t filter as DNA is fibrous and will clog filter device
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3
Q

Once you have got rid of particulates and aggregates what 3 ways can you get rid of nucleic acids?

A
  • PRECIPITATE WITH 1% PROTAMINE SULPHATE
  • Incubate with DNase
  • extract with sonication
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4
Q

What is considered the best way to remove nucleic acids?

A
  • sonication of proteins using shear chromosomes

- breaks open cells removing nucleic acids

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5
Q

Once you’ve removed nucleic acids, what 2 ways can you remove small molecules?

A
  • ultrafiltration/buffer exchange

- dialysis

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6
Q

How does BUFFER EXCHANGE/ULTRAFILTRATION work?

Removal or small molecules

A
  • put into centrifuge
  • this allows small particles through but not large ones
  • get rid of most small molecules if ran a few times
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7
Q

How does DIALYSIS work?

Removal or small molecules

A
  • put into solution, equilibrates so lets out small molecules but not proteins
  • keep replacing the buffer
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8
Q

You can purify your protein by putting it into E.Coli. What can happen though if you do this sometimes?

A
  • e.coli can produce a large amount of the protein if you put it into it so you will end up with too much protein
  • meaning you can’t fold them all at once
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9
Q

When purifying proteins, this is done so by exploiting physical and chemical properties. What are these 5 properties?

A

Charge - from sequence can work out charge
Hydrophobicity - most hydrophilic parts in centre, some on surface grouped together - cant predict where these are
Affinity - may have an idea if it binds metal ions
Solubility/Stability - depends on how strongly folded, and how large hydrophobic core, wouldn’t know unless you looked into this
Molecular Weight - need pure protein in order to calculate this

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10
Q

What way can you exploit solubility and what can this be used to purify?

A
  • purification by precipitation

- protein or contaminants

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11
Q

Do you want to increase or decrease solubility of the protein when trying to precipitate it?

A
  • Want to reduce solubility in order to precipitate other proteins
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12
Q

Precipitation SOLUBILITY

Changing ionic strength - What is salting in?

A
  • first stage of the salting in salting out process
  • low concentrations of salt is added to a protein solution
  • the solubility increases
  • Salt molecules stabilize protein
  • no precipitation of the proteins
  • increases solubility
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13
Q

Precipitation SOLUBILITY

Changing ionic strength - what is salting out?

A
  • hydrophobic patches order H2O around them form water cages which are entropically unstable
  • as salt conc increases from salting in concentration levels, H2O cages are removed exposing hydrophobic patches
  • therefore proteins aggregate and precipitate out of solution
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14
Q

Process of salting out - in practice. What is used in the process?

A

Fractionation experiment -

  • Add salt, centrifuge and separate precipitate, add salt/
  • process repeats
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15
Q

Do the conditions of salting out have to be exothermic or endothermic and why?

A
  • endothermic as you can’t have solution heating up
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16
Q

Precipitation SOLUBILITY

2) CHANGING SOLVENT - how does it work?

A
  • Fractionation
  • two charges on the protein, Q1 and Q2 on other.
  • the charges are separated by a distance called R.
  • They feel a force and that depends on what medium they’re sat in.
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17
Q

Precipitation SOLUBILITY

3) CHANGING pH - how does it work?

A
  • At a pH close to proteins pI, it will be uncharged
  • therefore may aggregate due to hydrophobic attraction
  • this is known as ‘isoelectric precipitation’.
  • may occur in dialysis
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18
Q

What can purification by exploiting stability only be used on and why?

A
  • contaminants/impurites only (not protein)

- as its irreversible so denature impurities/contaminants only

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19
Q

Is purification by exploiting proteins on solubility by selective denaturation reversible or irreversible?

A
  • IRREVERSIBLE
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20
Q

Apart from Solubility and Stability what do the other methods (CHAM) use?

A

Chromatography rather than fractionation/precipitation methods

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21
Q

What are the key features in chromatography which allow for it to work?

A
  • stationary phase
  • chromatographic bed eg column or glass or paper
  • mobile phase
  • delivery system
  • detection system
  • proteins separated by interaction with stationary phase
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22
Q

What is the stationary phase and how does it work?

A
  • stationary phase which is thing which will interact with protein (immobilised on matrix)
  • matrix: doesn’t interact, it is stable and inert.
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23
Q

What is high performance liquid chromatography?

A
  • instead of a solvent, proteins forced through under high pressures
  • much faster
  • Incompressible stationary phases
  • Continuous monitoring
24
Q

How does chromatography actually work/what occurs?

A
  • unbound proteins washed through
  • bound proteins are eluted by a change in condition
  • The peaks widen due to diffusion and retention on the column
  • This limits the resolution of peaks
25
Q

In chromatography resolution, what happens to the stationary phase as the column is ran?

A
  • Stationary phase holds onto some of the molecules, gets gradually wider the longer u run the column
26
Q

In protein clarification, what does filtration do and why is it not favourable?

A
  • filtration can lower protein yield as proteins stick and pores get clogged up
27
Q

In protein clarification, because filtration is not favourable, which method instead is used?

A
  • sedimentation is better
  • it involves centrifugation instead
  • on a large scale (supernatant soluble proteins) or a small scale (pellet: membrane fraction, organelles)
28
Q

What are the advantages of getting rid of nucleic acids by PRECIPITATING WITH 1% PROTAMINE SULPHATE?

A
  • simple

- rapid

29
Q

What are the disadvantages of getting rid of nucleic acids by PRECIPITATING WITH 1% PROTAMINE SULPHATE?

A
  • add contaminants sometimes

- poor reproducibility

30
Q

What conditions are required for Incubation with DNase to remove nucleic acids?

A
  • 30mins at 25-37 Degrees C
31
Q

What are the advantages of incubating proteins with DNase to remove nucleic acids?

A
  • simple

- reproducible

32
Q

What are the disadvantages of incubating proteins with DNase to remove nucleic acids?

A
  • slow

- proteases may degrade target

33
Q

In which conditions can sonication of proteins be performed in which the other 2 methods can’t be?

A
  • can be performed at low temperatures

- meaning it is faster

34
Q

What is solubility determined by?

A
  • how protein interacts with another protein that it meets
35
Q

In purification by precipitation (exploiting based on solubility) what charges do the proteins need?

A
  • Proteins need opposite charges, otherwise may repulse.
36
Q

How do you purify by solubility?

A
  • via precipitation
37
Q

What 3 things can you change to try and induce purification by precipitation (exploiting the solubility of the protein)?

A

1) Changing ionic strength - salting in and salting out
2) Changing the solvent
3) Changing the pH

38
Q

In salting out, what property of proteins make them salt out faster?

A
  • Proteins with more, larger patches precipitate faster
39
Q

What are the advantages of salting out?

A
  • pure
  • cheap
  • non interacting
  • Limits bacterial growth
  • Prevents denaturation
  • Concentration step
40
Q

What are the disadvantages of salting out?

A
  • Need to desalt (get rid of the salt)
  • Ineffectual if protein concentration too low
  • Results vary with extract components
  • can get co-precipitation
41
Q

How do you work out the force between the two protein charges Q1 and Q2?

A

KQ1KQ2 / dielectric constant x (r)squared

42
Q

The screening effect is measure by a dielectric constant in precipitation by changing solvent. What is the dielectric constant?

A
  • a factor explaining how permitive the medium is
43
Q

In the equation to work out the force between the two protein charges Q1 and Q2, you divide by dielectric constant. What is the dielectric constant of water and organic solvents?

A
  • Water ε=80

- organic solvents ε=1-10

44
Q

What is the effect of adding an organic solvent?

A
  • Adding organic solvents with lower dielectric constants increases electrostatic attraction
  • this causes selective precipitation
  • the organic solvent blocks the hydrophobic interactions meaning there’s more ionic interactions - so proteins precipitate.
45
Q

Precipitation SOLUBILITY
Changing the Solvent -
What happens when you add ethanol to the solution?

A
  • if you add ethanol to solution - lower solvating power

- so will precipitate out

46
Q

One disadvantage of adding ethanol…

A
  • may unfold protein
47
Q

When is it useful to use purification by exploiting stability?

A
  • useful if the protein of interest is particularly stable to e.g. Temperature, pH or solvent.
48
Q

How is the stationary phase prepared?

A
  • Poured into column trying not to get any air bubbles

- e.g. agarose (Sepharose), cellulose, dextran (sephadex)

49
Q

What does the slurry stationary phase work best on, and what sort of resolution does it produce?

A
  • larger particles, faster flow

- pourer resolution

50
Q

What does high performance liquid chromatography allow which normal solvent based chromatography doesn’t?

A
  • to use a smaller particle size for the column packing material
  • allows a much better separation of the components of the mixture
51
Q

In chromatography which proteins come off first?

A
  • proteins that’re weakly bound come off first

- strong come off later

52
Q

What can you do to the column to make separation of the proteins better in chromatography?

A
  • Make columns longer so proteins take longer to get out so they separate more
53
Q

What does the chromatographic bed do in chromatography?

A

holds the stationary phase

54
Q

What is the mobile phase and what is dissolved in it?

A
  • Liquid or gas passed over stationary phase

- Proteins are dissolved in this

55
Q

What does the delivery system do in chromatography?

A

Adds proteins to the column

56
Q

How do you work out the resolution in chromatography using the peaks?

A

Resolution = 2S/(w1+w2)

BB10186 (4 decks)

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