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BB10186 > Lec 3 and 4 Crennell > Flashcards

Lec 3 and 4 Crennell Flashcards

1
Q

SEPERATION BY CHARGE

How does Ion exchange chromatography work in an anion exchanger?

A
  • at pH above a protein’s pI it has overall negative charge and will bind to a matrix that is positively charged
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2
Q

What conditions are required for adsorption in ion exchange chromatography?

A
  • Low ionic strength buffer

- pH to generate opposite charge to ion exchange resin

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3
Q

What is pI?

A
  • The pH at which the net charge on the protein is zero
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4
Q

What is Elution?

A

elution is the process of extracting one material from another by washing with a solvent;

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5
Q

When eluting with salts in IEC, what is very important that you don’t do?

A
  • Important that you don’t change the pH
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6
Q

What does focusing do in IEC?

A
  • Focussing increases resolution
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7
Q

What are the advantages of IEC Elution Column?

A
  • Load large volume, elute small, Concentrating

- High capacity, good resolution

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8
Q

What are the disadvantages of IEC Elution Column?

A
  • Denaturation at pH extremes
  • Product in high salt
  • pH gradient technically difficult
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9
Q

How does Hydrophobic Interaction chromatography work?

A
  • SALTING OUT
  • Proteins loaded in a high ionic strength buffer bind to a hydrophobic column.
  • Protein have areas that are polar non polar and hydrophobic etc
  • Remove from column by lowering the amount of salt (due to water cages)
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10
Q

What order do you elute in? (hydrophobicity)

A
  • Elute in order of hydrophobicity

- Most hydrophobic last.

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11
Q

What are the 4 steps in hydrophobicity elution HIC?

A

1) Equilibration
2) Sample application
3) Gradient Elution
4) Regeneration

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12
Q

What are the limitations of Hydrophobic Elution HIC?

A
  • some proteins may not bind as column isn’t sufficiently hydrophobic
  • some may bind too well and not come off
  • too little salt then the water cages will remain and therefore the proteins wont bind
  • could bind to each other
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13
Q

In HIC you can interfer with protein interaction to the column, how may you do so?

A
  • decreasing salt concentration
  • changing pH
  • changing temperature
  • adding detergent
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14
Q

How does Affinity chromatography work?

A
  • Proteins interact strongly with a substrate or binding partner.
  • Ligand is attached to an inert matrix binds only the desired protein
  • Interaction must be sufficiently strong to allow attachment, but weak enough to allow detachment
  • To elute you add a free ligand, or change pH or ionic strength.
  • Affinity compound attached to inert matrix
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15
Q

Measuring Affinity (Affinity Chromatography)

A

Affinity Ka = [PL] / [P]x[L] (tendency to associate)

  • dissociation is the opposite Kd
  • Both specific but Process must be reversible so that protein can bind with ligand but also break off
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16
Q

What is an example of Affinity purification?

A
  • Staphylococcal nuclease

- binds base analogue, blue allows it to attach to matrix, eluted by pH

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17
Q

What considerations must you take into account for Affinity Chromatography?

A
  • consider protein stability within conditions using
  • Means of attaching ligand to matrix needs to be sufficiently stable so that it doesn’t come off in the reaction
  • Elute, must change Kd by 10 fold in order to switch from binding to not binding, do so by adding more ligand and eq will shift from being bound to matrx to free ligand
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18
Q

What are some advantages of Affinity Chromatography?

A
  • > 95% purity in one step

- Add tags which can help protein fold properly

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19
Q

What is the Only disadvantage of Affinity Chromatography?

A
  • If you haven’t used recomb tags, finding specific place where ligands attach can be hard (only disadvantage) very good usually
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20
Q

How does Size exclusion (gel filtration) Chromatography work?

A
  • Separation by size (assuming all proteins have same shape)
  • Solid matrix with pores
  • Proteins of diff size travel down, some can get into the pores depending on their size
  • separated based on that
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21
Q

What are the key features of gel filtration (size exclusion)?

A
  • Non-adsorptive
  • gentle
  • elute in buffer used to run column (desalting)
  • no gradient required
  • can use whichever conditions your protein needs
  • cross-linked, open 3D matrix
  • pores of variable sizes
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22
Q

How do you calculate molecular weight in (Size Exclusion Chromatography)?

A 
- Proportion of pores occupied by a protein =
Kav = (Ve -V0) / (Vt - V0)
- V0 = void volume (outside the beads)
- Vt - total volume 
- Ve = excluded volume of a protein
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23
Q

In a graph of Kav what is Kav proportional to?

A
  • Kav proportional to log (MW)
  • calibration with known MW
  • Line is straight between 0.1 and 0.7
  • Curves above and below those values
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24
Q

How would you separate the peaks better in gel filtration?

A
  • decrease peak width to separate peaks better
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25
Q

What are the advantages of Size exclusion chromatography gel filtration?

A
  • Experimentally simple
  • Gentle, conditions as required by protein
  • can desalt
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26
Q

What are the disadvantages of Size exclusion chromatography gel filtration?

A
  • Long Columns
  • Time is long
  • Small samples
  • Diluting required
  • Just a polishing step
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27
Q

What are the overall Aims of protein purification?

A
  • By end trying to get rid of every other contaminant
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28
Q

Which techniques have high capacity?

A
  • Ion exchange

- affinity

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29
Q

Which techniques have low capacity?

A
  • size exclusion / gel filtration has low capacity
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30
Q

If 95% efficient after 8 steps then how much % of protein will you have?

A

80%

31
Q

Which technique is usually used first?

A

Affinity

32
Q

Which technique is usually used last as polishing step?

A
  • Size exclusion / gel filtration
33
Q

Which technique can be used at any stage but only once?

A
  • Ion Exchange
34
Q

What are the overall Stages of Purifying a protein?

A

1) Capture: High capacity
Precipitation (or IE) end: high ionic strength
2) Intermediate chromatographic steps, HIC start: high salt, end: low salt
IE start: low salt, end: high salt, concentrated
3) Polishing
GF start: concentrated, end: dilute
- Can desalt (fast GF, dialysis) or concentrate, but adds steps

35
Q

How can you detect impurities?

A

SDS-PAGE

IEF/Western blot/MS

36
Q

What is proportional in ion exchange chromatography?

A
  • strength is proportional to the net charge
37
Q

SEPERATION BY CHARGE

How does Ion exchange chromatography work in an cation exchanger?

A
  • below the pI, the protein will bind to a cation exchanger (negatively charged matrix).
38
Q

What property of your protein must you know in IEC?

A

pI

39
Q

In IEC if the pH is less than the pI what charge will the protein have?

A

positively charged

40
Q

What happens as pH increases in Ionic Exchange Chromatography?

A
  • As pH ↑ acidic amino acids deprotonate
41
Q

If pI is 8.5 (in cation exchanger) what pH are you likely to chose?

A

chose pH which is one pH unit away from protein if 8.5 could use 7.5 and 9.5

42
Q

Examples of groups attached in matrix in anion exchange chromatography

A

Weak - DEAE

Strong - Quaternary ammonium ‘Q’

43
Q

Examples of groups attached in cation exchange chromatography

A

Weak - Carboxymethyl

Strong - Methyl sulphonate ‘S’

44
Q

How does Ion exchange chromatography work? (SEP BY CHARGE)

A
  • When ionic strength is increased, proteins elute in order of net charge.
  • The pH is crucial and may require several trials to optimise.
  • Proteins more neg charge bind more strongly so will come off later
  • Lowest charge come off first from column
  • Most strongly bound need a lot of salt, either sodium or chloride depending on what matrix you have, - they compete with protein for the charges on the stationary phase
45
Q

What is chromatofocusing? (IEC)

A

Elution by pH gradient

46
Q

Proteins with different ???? separate as they pass through the column? IEC

A

diff pI’s

47
Q

What is the charge gradient in IEC?

A
  • positive to neutral to neg down column
48
Q

In low salt in the Hydrophobic Interaction Chromatography, what do the hydrophobic bits on the proteins have around them?

A
  • cages of water
49
Q

In high salt in the Hydrophobic Interaction Chromatography, what do the hydrophobic bits on the proteins do?

A
  • they don’t have H2O cages so they interact with the column
50
Q

Which proteins come off last in HIC?

A
  • Most hydrophobic proteins come off last
51
Q

How can you vary separation in HIC in the column?

A
  • changing the ligands (either aliphatic or aromatic molecules)
  • or the initial ionic strength
52
Q

In affinity chromatography, what may be an affinity property of the protein which you could exploit?

A
  • binding site of protein
53
Q

How many steps is Affinity Chromatography?

A
  • One step elution.
54
Q

What properties must the ligands have in the affinity chromatography column?

A
  • specific to a single protein
  • or to a group of proteins
  • must be attached without losing affinity. 
55
Q

How is Affinity chromatography exploited using affinity tags?

A
  • recombinant addition of affinity tags to proteins to aid purification
  • examples of tags - could be proteins (e.g. maltose-binding protein) or smaller tags e.g His6.
56
Q

How is Affinity chromatography exploited using affinity tags?

A
  • recombinant addition of affinity tags to proteins to aid purification
57
Q

Examples of tags used in affinity chromatography…

A

could be proteins (e.g. maltose-binding protein) or smaller tags e.g His6.

58
Q

How might you elute in Affinity chromatography?

A
  • by changing pH or salt, salt disrupts salt or polar hydrophobic interactions
59
Q

Which sized proteins come off first in gel filtration?

A
  • largest proteins come off first

- smallest go into all the pores

60
Q

What is the separation like using smaller beads in gel filtration? (size)

A
  • smaller beads - most proteins squashed up at high molecular weight end
61
Q

What is the separation like using medium beads in gel filtration? (size)

A
  • medium beads have good separation of proteins
62
Q

What is the separation like using larger beads in gel filtration? (size)

A
  • large beads all come off late and close together
63
Q

What order of techniques based on their capacity/resolution would you use in overall protein purification?

A
  • At start, have a lot of proteins, go from techniques with high to low capacity
  • Want to go from techniques with increasingly higher resolution
64
Q

Which two ways can you elute in IEC

A
  • elute by adding salts,

- elute by changing the pH

65
Q

Elution by salt in IEC - how does it work?

A
  • proteins come off in order of net charge
  • add salt (+or- depending on cation or anion) surrounds the protein, keep adding salt, salt surrounds charges on protein so it cant bind to stationary phase
  • so then eluted out of column
  • step wise gradient
66
Q

What are the pros of an activity assay?

A

Pros
Tailored to protein
Rapid, reproducible, specific, cheap, sensitive

67
Q

What are the cons of an activity assay?

A
  • Often colourimetric
  • destructive
  • reduces yield
68
Q

When you monitor purity via Absorption at 280nm of the total protein, what are the pros?

A

Rapid, non destructive

69
Q

When you monitor purity via Absorption at 280nm of the total protein, what are the cons?

A

Not quantitative (e unknown), nucleic acids interfere

70
Q

When you monitor purity via Bradford assay of the total protein, what are the pros?

A

Simple, rapid, fewer interfering substances

71
Q

When you monitor purity via Bradford assay of the total protein, what are the cons?

A
  • Variation in response

- requires standard curve

72
Q

In a purification table how do you work out Specific activity?

A

Specific activity = Enzyme activity/unit mass protein

73
Q

In a purification table how do you work out Yield?

A

Total activity after nth step x 100% / Total enzyme activity in initial sample

74
Q

In a purification table how do you work out Purification factor?

A

Specific activity after the nth step / Specific activity of initial sample

BB10186 (4 decks)

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