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BB10186: Proteins purification and characterisation > lecture 4 > Flashcards

lecture 4 Flashcards

1
Q

What is the ligand in regards to affinity chromatography?

A
  • Attached to an inert matrix , binds only the desired proteins
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2
Q

How can we elute the the protein from affinity chromatography?

A

Interaction must be disrupted by adding the free ligand or by changing pH of ionic strength

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3
Q

What is the matrix in affinity chromatography?

A

Its an inert matrix, with an affinity compound attached

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4
Q

What are the 3 different types of biospecific ligand?

A
  • specific
  • group specific
  • pseudo specific
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5
Q

How can you measure affinity?

A

Ka and kd are the inverse of each other

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6
Q

What is an example of affinity purification?

A
  • staphylococcal nuclease
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7
Q

How is Staphylococcal nuclease purified?

A
  • binds base analogue

- eluted by change in pH

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8
Q

What are the 3 things you must consider when doing affinity chromatography ?

A
  • protein stability
  • Ligand attachment preserves affinity
  • elution
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9
Q

What is the ligand modification in affinity chromatography?

A

spacer to increase accessibility

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10
Q

How can we elute from affinity chromatography?

A
  • use a free ligand
  • pH
  • Increasing salt concentration
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11
Q

Why would we use affinity tags?

A

When a protein/ enzyme doesn’t have its own affinity , we can create that affinity using a tag

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12
Q

What is an example of an affinity tag?

A

HIs tag

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13
Q

What is immobilised metal affinity chromatography?

A

proteins separated accordingly to their affinity for metal ions that have been immobilised by chelators to an insoluble matrix

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14
Q

What is the normal process for immobilised metal affinity chromatography?

A
  • metal associates and protein with a HIs tag binds , elute with imidazole
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15
Q

What are the advantages of affinity chromatography?

A

> 95% purity in one step
Tagged recombinant proteins
(His)n
Proteins promote solubility

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16
Q

What is a disadvantage of affinity chromatography?

A
  • Findig an attachable specific ligand
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17
Q

How does gel filtration work?

A
  • forms pores of variable sizes which large proteins are unable to enter .
  • Thus elute the larger proteins first
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18
Q

What are the steps needed to perform gel filtration?

A
  • equilibration
  • sample application
  • elution
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19
Q

How do the proteins elute in gel filtration?

A
  • samples elute in decreasing size, largest comes off first
20
Q

What is the equation to determine the proportion of pores occupied by a protein?

A

Kav = (Ve- Vo)/ (Vt- Vo)

21
Q

What does Vo stand for?

A

Vo = void volume (outside the beads)

22
Q

What does Vt stand for?

A

Vt = total volume

23
Q

What does Ve stand for?

A

Ve = excluded volume of a protein

24
Q

How is the Kav related to the molecular weight of the protein?

A

Kav proportional to log (MW)

Calibration with known MW

25
Q

How can you ensure separation of peaks of proteins in gel filtration ?

A
  • range of bead sizes
  • increasing the column length
  • peak width
26
Q

What are the advantages of gel filtration?

A
  • simple
  • Gentle, conditions as required by protein
    Complexes, oligomers  Characterisation
  • Desalting
27
Q

What are the disadvantages of gel filtration?

A
  • Long columns, time

- Small samples, diluting

28
Q

Which techniques have a high capacity?

A

ion exchange, Affinity chromatography and precipitation

29
Q

Which techniques how a small resolution?

A

precipitation

30
Q

Which techniques have a small capacity

A

gel filtration

31
Q

Which techniques have a high resolution?

A
  • Ion exchange , Affinity chromatography and Gel filtration
32
Q

What is the overall best strategy for purifying proteins?

A

Capture: High capacity
- Precipitation (or IE) end: high ionic strength
Intermediate chromatographic steps
HIC start: high salt, end: low salt
IE start: low salt, end: high salt, concentrated
Polishing
GF start: concentrated, end: dilute

33
Q

What are the steps to isolate Deacetoxycephalosporin C synthase?

A
  1. E.coli lysed by sonication
  2. Centrifuge ro clarify
  3. Ion exchange : gradient vs stepwise
  4. HIC
  5. GF to polish
34
Q

What are the steps used to isolate MBP - tagged protein =?

A
  1. Maltose - bound column
  2. Gel filtration
  3. Analysis y SDS page
35
Q

What are the steps used to isolate FAB fragment?

A
  1. cells lysed with sucrose
  2. DNAase added
  3. cation exchange
  4. HIC pH 5 start 1M (NH4)2SO4
    Step to 50%
  5. IE with shallow gradient
36
Q

What are the 3 ways we can monitor purity?

A
  • specific assays
  • total protein concentration
  • purification table
37
Q

What are the 3 types of assays for determinng the purity?

A
  • activity assay
  • binding assay
  • detection of impurities
38
Q

What are the advantages of an activity assay?

A
  • tailored to protein

- rapid , reproducible , specific cheap

39
Q

What are the disadvantages of an activity assay?

A

often colorimetric , destructive and reduces yield

40
Q

What are the types of detection of impurities that can be used?

A

SDS-PAGE

IEF/Western blot/MS

41
Q

What are the types of monitoring where the total protein is detected?

A
  • absorption at 280 nm

- Bradford assay

42
Q

Pros vs cons of. the absorption at 280 nm?

A

pros - Rapid, non destructive

cons - Not quantitative (e unknown), nucleic acids interfere

43
Q

What are the pros and cons of the Bradford assay?

A

pros Simple, rapid, fewer interfering substances

cons -Variation in response, requires standard curve (is actually a straight line)

44
Q

Equation for specific activity

A

Enzyme activity/unit mass protein

45
Q

What is the equation for yield?

A

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

46
Q

What is the equation for the purification factor?

A

Specific activity after the nth step

/ Specific activity of initial sample

BB10186: Proteins purification and characterisation (10 decks)

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