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BB10186 > lecture 3 > Flashcards

lecture 3 Flashcards

1
Q

Chromatography can be applied to which elements within chasm

A

1- charge
2- hydrophobicity
3- Affinity
4- Molecular weight

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2
Q

Ion exchange theory works on the basis of?

A

Overall charge can be determined if we know the sequence

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3
Q

Surface charge vs overall protein charge?

A

may be different

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4
Q

What type of interaction is occurring in ion exchange chromatography?

A

electrostatic

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5
Q

Matrix within Ion exchange chromatography contains

A

ionised groups

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6
Q

Anion exchange therefore?

A

exchanges anions
matrix- pos
proteins-neg

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7
Q

Cations charge

A

cats pur positive

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8
Q

Strength of the interaction between the ionised groups and the protein is due to?

A

number of ionic charges the proteins have

separation due to charge

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9
Q

Matrix functional group examples

A 
Anion exchanger
- Diethylamino ethyl (DEAE)
- Quaternary ammonium 
Cation exchanger
- Carboxymethyl 
- Methyl sulphonate
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10
Q

Stronger anions and cations

A

will interact over a larger range

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11
Q

issue with really strong or really weak binders?

A

will bind too much or not enough

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12
Q

Issue with anion and cation exchanges

A

may not be the appropriate pH

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13
Q

Conditions for adsorption?

A

Low ionic strength buffer

pH to generate opposite charge to ion exchange resin (know pI)

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14
Q

pH

A

protonated (positively charged)

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15
Q

pI for a cation exchanger

A
  • high (neg can bind to positive)
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16
Q

How to choose buffer pH

A

one pH unit away from the pI

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17
Q

Choose a suitable buffer pH for a cation exchanger when the pI=8.5

A

Can choose 1 pH unit above or bellow

  • 9.5- too high (alkali)
  • 7.5 better due to this being a cation exchanger (therefore your ions will be pos charged)
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18
Q

Elution method ?

A

change in ionic strength

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19
Q

Highly charged ions will come off?- elution

A

last

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20
Q

What type of gradient is drown in elution

A

stepwise

gradient

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21
Q

What needs to be maintained during elution

A

pH as this may change the proteins ability to bind

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22
Q

Choose gradient such that?

A

they ate separated

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23
Q

Elution by changing the pH

A

Molecules with the same isoelectric point are focused in narrow bands during the separation.

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24
Q

What type of gradient in the collum- Elution by changing the pH. Usually is ?

A

pH gradient

usually low to high

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25
Q

Focussing increases?

A

resolution

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26
Q

when might we want to use the focussing technique to separates proteins?- range ?

A

isoforms
eg haemoglobin
usually over a range of 1 and a Half pH unit

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27
Q

Advantages of ion exchange chromatography?

A

1- Load large volumes, elute small (concentrating)

2- High capacity, good resolution

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28
Q

Disadvantages of ion exchange chromatography?

A

denaturation at extreme pH
product in high salt
pH gradient technically difficult to produce

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29
Q

Seperation due to?- Hydrophobic interaction chromatography

A

differing surface hydrophobicity

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30
Q

Hydrophobic interaction chromatography promoted by?

A

Salting out

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31
Q

In low salt?

A

water cages form around hydrophobic patches

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32
Q

The most hydrophobic will elute?

A

last

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33
Q

In high salt?- hydrophobicity

A

Water cages removed, bind to ions

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34
Q

how might we disrupt the interaction between the protein and the column- elution?

A
  • reducing salt
  • changing salt type
  • adding detergent
  • changing pH
  • reduce temperature
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35
Q

Affinity chromatography relies on?

A

Isolate using specific property of protein

e.g. binding site

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36
Q

Affinity compound attaches to ?

elution and purification from this method?

A

inert matrix
binds only the seared protein
one step elution
3 fold purification

37
Q

by what bonds are ligands attached to the matrix?

A

covalently

38
Q

Ligands in affinity chromatography are _____specific

A

bio

39
Q

Examples of molecules raised against proteins - affinity

A
  • substrate enzyme
  • inhibitor
  • cofactor
  • antibodies
40
Q

Group specific examples?

A

Protein A or G (antibody)

Lectins (glycoproteins)

41
Q

Pseudo specific

A

Dyes mimic cofactors

42
Q

affinity equation?

A

Ka= [PL]/[P][L]

P+ L»> PL

43
Q

Dissociation?

A

Kd= [P][L]/[PL]

44
Q

Kd value in which is strong enough to adsorb

” to elute

A

Kd<10^-4

Kd>10^-8

45
Q

Staphylococcal nuclease example

A

Binds to analogue

Eluted by changing the pH, therefore no longer charged and is washed off

46
Q

How does protein stability need to be considered in affinity chromatography?

A

‘Spacer arm’ needed to increase the distance from the interaction area.

To prevent delocalisation during purification (preserves affinity)

47
Q

Methods of elution- Affinity

A
  • large concentrations free ligands (not bound to the matrix) - cost
  • pH
  • Salt (detergent)
48
Q

Affinity tags usually bind to ??

A

Metal ions within the matrix

49
Q

Affinity tags are tagged in what sense ??

A

tagged recombinantly (using a vector to produce)

50
Q

examples of affinity tags

A

maltose-binding protein

- or (His)n (where n≥6)

51
Q

within the matrix what is present?- affinity tags, EXAMPLES

A

Metal associates, part of coordination sphere

Ni2+- example

52
Q

(his)n tag binds with?

A

imidazole

strongly interacts with the ni which causes the protein of interest with the his tag to dissociate

53
Q

Advantages vs disadvantages of affinity chromatography

A

> 95% purity in one step
- Tagged recombinant, proteins promote solubility

disadvantages:
- finding attachable specific ligand

54
Q

By adding larger tags

A

help correct protein folding

55
Q

gel filtration assumes?

A

all proteins have the same shape and therefore are separated only due to their size

56
Q

in the case of gel filtration what type of matrix ?

A

solid matrix

pourous matrix

57
Q

conditions in gel filtration?

A

same conditions

58
Q

characteristics of gel filtration?

A

Non-adsorptive, gentle

59
Q

Proportion of pore occupied by a protein?

A 
Kav = (Ve- Vo)
         (Vt - Vo)
Ve- excluded vol of protein 
Vo= void volume
Vt- total volume
60
Q

Kav us proportional to?

A

log (mw)

61
Q

proteins come off in??- GF

A

voids

small come off last

62
Q

beads must be able to ____ ___ _______ therefore ?

A

encompass the protein, therefore there are a range of bead sizes and materials

63
Q

Peak width is due to ?

A

size of beads

speed of flow

64
Q

advantages and disadvantages of gel filtration?

A
  • simple
  • gentle (conditions as required by the protein)
  • desalting
dis
- long columns
- Time consuming 
- small samples 
diluting
65
Q

Gel filtration is often referred to as a?

A

polishing step

66
Q

The matrix is usable composed of

A

commonly agarose, cellulose or dextran,
is milled into beads of various sizes and porosity,
with different optimal separation ranges.

67
Q

Maximise the final yield?

A

Minimise the step number

68
Q

Low capacity vs high capacity methods?

A

low cap:
Gel filtration

High cap:
Ion exchange
Affinity chromatography
salting out (ammonium sulphate)

69
Q

High resolution methods vs low resolution methods

A

high res:
Ion exchange
Affinity chromatography
Gel filtration

low resolution: salting out (ammonium sulphate)

70
Q

detection of impurities

A
  • SDS-PAGE

- IEF/Western blot/MS

71
Q

Deacetoxycephalosporin c synthase method of protein purification?

A
  • Lysed by sonication
    2. centrifuge to clarify
    3. Ion exchange
    4. hydrophobic interaction (add low salt
    5. Gel filtration
72
Q

MBP-tagged protein 3 steps to purifications

A
  1. Maltose- bound column
  2. Gel filtration
  3. SDS-PAGE analysis
73
Q

Fab fragment example

A
  1. cell lysed with sucrose
  2. DNAse added
  3. Cation exchange
  4. salting out
  5. IE with shallow gradient
74
Q

recombinant myoglobin ?

A

express in ecoli
soluble fraction removed
affinity chromotograohy
size exclusion chromatography

75
Q

Activity assays are ______ to your protein
pros
cons

A

Tailored

pros: rapid, reproducible, specific, cheap, sensitive
cons: colourmetrix, destructive and reduces yield

76
Q

Specific assay examples? general names given

A
  • activity assay
  • binding assay
  • detection of impurities
77
Q

Monitor purification two method names?

A
  • adsorption at 280nm

- Bradford assay

78
Q

Concentration of protein needed for absorption at 280nm method?

A
  • 0.1 mg/ml
79
Q

absorption at 280nm method

PROS AND CONS

A 
PROS: rapid and non destructive 
non quantitve (e unknown) and nucleic acids interfere
80
Q

Bradford assay range of volumes

A

1-2 ul

81
Q

pros and cons of the Bradford assay?

A

pros: simple, rapid. fewer interferon sustances
cons: Variation In response, requires a calibration curve

82
Q

Specific activity equation?

A

= Enzyme activity/ unit mass protein

83
Q

yield=

A

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

84
Q

purification factor equation?

A

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

85
Q

recombinant myoglobin analogy names

A

ELIO LIKES CUMING AND SCREMING

Ecoli expression

  • LYSE
  • CENTRIFUGE
  • affinity
  • Size exclusion
86
Q

Deacetoxycephalosporin c synthase analogue

A

STOP CUMMING IN HER GAF

  • Sonnication (ecoli)
  • Centrifuge
  • ion exchangge
  • hydrophobicicty chrom
  • Gel filtration
87
Q

MBP -tagged protein analogue

A

Maxine Gee sexy

  • Maltose binding column
  • Gel filt
  • SDS (analysing)
88
Q

FAB Fragment analogue ?

A

llamas drive cars singing immensely

  • Lysed (sucrose)
  • Dnase
  • Cation exchanger
  • Salting (wash with low salt)
  • IE (shallow gradient)
89
Q

Bradford assay?

A

The Bradford protein assay is used to measure the concentration of total protein in a sample

BB10186 (6 decks)

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