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BB10186: Proteins purification and characterisation > lecture 4 > Flashcards

lecture 4 Flashcards

(46 cards)

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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?

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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?

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A

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

21
Q

What does Vo stand for?

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A

Vo = void volume (outside the beads)

22
Q

What does Vt stand for?

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A

Vt = total volume

23
Q

What does Ve stand for?

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A

Ve = excluded volume of a protein

24
Q

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

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A

Kav proportional to log (MW)

Calibration with known MW

25
How can you ensure separation of peaks of proteins in gel filtration ?
- range of bead sizes - increasing the column length - peak width
26
What are the advantages of gel filtration?
- simple - Gentle, conditions as required by protein Complexes, oligomers  Characterisation - Desalting
27
What are the disadvantages of gel filtration?
- Long columns, time | - Small samples, diluting
28
Which techniques have a high capacity?
ion exchange, Affinity chromatography and precipitation
29
Which techniques how a small resolution?
precipitation
30
Which techniques have a small capacity
gel filtration
31
Which techniques have a high resolution?
- Ion exchange , Affinity chromatography and Gel filtration
32
What is the overall best strategy for purifying proteins?
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
What are the steps to isolate Deacetoxycephalosporin C synthase?
1. E.coli lysed by sonication 2. Centrifuge ro clarify 3. Ion exchange : gradient vs stepwise 4. HIC 5. GF to polish
34
What are the steps used to isolate MBP - tagged protein =?
1. Maltose - bound column 2. Gel filtration 3. Analysis y SDS page
35
What are the steps used to isolate FAB fragment?
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
What are the 3 ways we can monitor purity?
- specific assays - total protein concentration - purification table
37
What are the 3 types of assays for determinng the purity?
- activity assay - binding assay - detection of impurities
38
What are the advantages of an activity assay?
- tailored to protein | - rapid , reproducible , specific cheap
39
What are the disadvantages of an activity assay?
often colorimetric , destructive and reduces yield
40
What are the types of detection of impurities that can be used?
SDS-PAGE | IEF/Western blot/MS
41
What are the types of monitoring where the total protein is detected?
- absorption at 280 nm | - Bradford assay
42
Pros vs cons of. the absorption at 280 nm?
pros - Rapid, non destructive cons - Not quantitative (e unknown), nucleic acids interfere
43
What are the pros and cons of the Bradford assay?
pros Simple, rapid, fewer interfering substances cons -Variation in response, requires standard curve (is actually a straight line)
44
Equation for specific activity
Enzyme activity/unit mass protein
45
What is the equation for yield?
Total activity after nth step x 100%/ Total enzyme activity in initial sample
46
What is the equation for the purification factor?
Specific activity after the nth step | / Specific activity of initial sample

BB10186: Proteins purification and characterisation (10 decks)

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