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Proteins > Protein Purification and Characterisation > Flashcards

Protein Purification and Characterisation Flashcards

1
Q

Give 7 applications of Gel Filtration.

A
  1. Buffer exchange
  2. Desalting, removal of small molecules
  3. Purification of proteins based on their mass
  4. Identification of quaternary arrangements (formation of dimer, tetramer, etc.)
  5. Analysis of polydispersity of purified samples
  6. Separation of protein complexes from unassembled individual components
  7. Removal of undersired aggregation products of very high molecular weight (void volume)
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2
Q

Gel filtration chromatography is also known as

A

size exclusion chromatography (SEC)

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3
Q

SEC is a method of chromatography which is ________

A

non-adsorption

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4
Q

SEC separates proteins based on their _______ radius

A

hydrodynamic

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5
Q

What is Stokes diameter?

A

the diameter of the sphere that molecules occupy as they tumble in solution

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6
Q

In SEC, do larger or smaller molecules fall through the column faster? Explain why

A

small molecules take longer to fall through the column, because they get trapped in the pores of the cross-linked polymer beads (gel matrix). The large molecules are too big to enter the pores of the beads and so flow through the buffer solution quickly.

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7
Q

Give the equation linking: Kd, Vi, Ve and V0

A

Kd = (Ve-V0)/Vi

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8
Q

for the equation Kd=(Ve-V0)/Vi, what does V0 denote?

A

V0 = Void volume. It is the free accessible area, fully accessible for all molecules.

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9
Q

for the equation Kd=(Ve-V0)/Vi, what does Vi denote?

A

Vi = Inner volume. It is the aqueous space between the beads i.e. the pores. IT is accessible for small molecules and solutes.

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10
Q

For most molecules, what shape would the graph be for Kd v logMw following SEC?

A

straight line graph with negative gradient

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11
Q

Which gel matrices can be used for desalting?

A

sephadex (dextran) or biogel (polyacrylamide)

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12
Q

Which gel matrices can be used for peptide separation?

A

Sephadex (dextran) or Biogel (polyacrylamide)

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13
Q

Which gel matrices can be used for peptide fractionation?

A

Sephadex (dextran), Sepharose (agragose), or biogel (polyacrylamide)

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14
Q

Outline 4 disadvantages of SEC.

A
  1. performance is very sensitive to column packing
  2. can have non-specific interactions between protein and resin, which decreases resolution
  3. offers low resolution for complex protein mixtures
  4. sample must be loaded at small volume for adequate resolution. This can be problematic for proteins that precipitate at high concentrations.
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15
Q

IEC is a form of _______ chromatography

A

adsorption

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16
Q

what are the 2 types of IEC

A

cation and anion exchange

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17
Q

For IEC, a _____ column is usually used

A

short

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18
Q

IEC separates proteins according to their ______________

A

net surface charge

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19
Q

Name the 7 possible functions of proteins.

A 
Catalytic
Binding
Transport
Structural
Motor
Storage
Signalling
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20
Q

Give an example of a catalytic protein

A

alcohol dehydrogenase

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21
Q

Give an example of a binding protein

A

antibody

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22
Q

Give an example of a transport protein

A

haemoglobin

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23
Q

Give an example of a structural protein

A

collagen/keratin/cytoskeleton proteins

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24
Q

Give an example of storage protein

A

Ferritin

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25
Q

Give an example of a signalling protein

A

insulin

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26
Q

What is the purpose of a binding protein

A

binds ligands and releases them at target locations

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27
Q

Give an example of a motor protein

A

actin, dynein, myosin

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28
Q

Determination of purity

A

SDS page, isoelectric focusing, peptide mapping

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29
Q

Chemical and mass characterisation

A

N-terminal sequencing, acid analysis, mass spec, SDS page

30
Q

Conformational analysis

A

Circular dictorism, fluorescence spectroscopy, NMR, X-ray diffraction

31
Q

Analysis of quaternary structure

A

gel filtration, native gel electrophoresis, analytical centrifugation, light scattering NMR, X-ray differaction

32
Q

SDS Page can be used for

A

Determination of purity, chemical and mass characterisation

33
Q

NMR can be used for

A

conformational analysis, analysis of quaternary structure

34
Q

X-ray diffraction can be used for

A

conformational analysis, analysis of quaternary structure

35
Q

What are the two broad categories of column chromatography?

A

Non-adsorption and adsorption

36
Q

outline what is meant by non-adsorption in column chromatography

A 
gel filtration (size exclusion)
proteins distributed between the two phases, travelling at different speeds
long column required
37
Q

outline what occurs in adsorption column chromatography

A

ion exchange;
short column used.
Attachment depends on the polarity of the solutes i.e. the affinity for the solid phase.
If the stationary phase is more polar than the mobile phase then high polar compounds in the mixture will tightly bound to the stationary phase. Less polar compounds will lightly bind to the stationary phase. Less tightly bound compounds will be eluted out by the mobile phase earlier than the tightly bonded ones.

38
Q

In SEC, what is a gel matrix?

A

cross linked polymers with pores acting as molecular sieves. Stationary phase

39
Q

Name 3 cation exchangers

A

sulphonopropyl
methyl sulphonate
carbonyl methyl

40
Q

name 3 anion exchangers

A

quaternary ammonia
diethylaminoethyl
quaternary aminoethyl

41
Q

ion exchange chromatography separates proteins according to their ability to ______

A

adsorb the matrix based on their net surface charge

42
Q

Name the 4 steps for IEC

A

equilibration
sample application and wash
selective elution
regeneration

43
Q

Isoelectric point (pI)

A

the pH when the net charge of the molecule is zero

44
Q

Retention volume (Vr) =

A

flow rate (F) x retention time (tR)

45
Q

how do you calculate electrophoresis velocity

A

velocity = electric force / friction force

46
Q

Polyacrylamide gel electrophoresis (PAGE) separates proteins according to ______

A

size

47
Q

SDS PAGE separates proteins according to ______

A

molecular weight

48
Q

Why do you heat up protein prior to SDS PAGE

A

to denature the protein; smaller proteins move further through the gel

49
Q

What are the components for SDS PAGE?

A

SDS
DTT
Glycerol
Bromophenol blue

50
Q

what is the function of SDS in SDS PAGE?

A

it’s an ionic and hydrophobic detergent which denatures proteins.
It induces a negative charge.
All polypeptide chains have similar conformations

51
Q

what is the function of DTT in SDS PAGE?

A

it reduces disulphide bonds
further denatures protein by overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure

52
Q

what is the function of glycerol in SDS PAGE?

A

increases density - makes bonds sharper

53
Q

what is the function of bromophenol blue in SDS PAGE

A

dye front

54
Q

in SDS PAGE, dyes and proteins are loaded into the stacking gel. Stacking gels have a ____ percentage of polyacrylamide, meaning there are _____ pores in this section. It has a ____ pH, and migration in is based on ______.
The proteins and dye then move into the separating/resolving gel. Here, there is a ____ percentage of polyacrylamide, meaning there are ____ pores. This gel will have a _____ pH, and migration here is also based on _____, but small proteins will move _____ through the gel than large proteins.

A

low
large
low
charge

high
small
high
charge
faster
55
Q

what is the most common dye for detection in SDS PAGE?

A

coomassie brilliant blue - binds non-specifically to all proteins, can be used qualitatively to determine concentration

56
Q

what is the most sensitive method of detection in SDS PAGE?

A

Silver stain -

It’s based on the reduction of Ag+ –> Ag

57
Q

What is the most specific method of detection for SDS PAGE?

A

Western blot - highly sensitive + specific.

Transfers proteins onto membrane. Sensitivity depends on specific antibody. Mw marker is used.

58
Q

Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their __________ along a continuous _____ gradient.

A

isoelectric point

pH

59
Q

at pH 7, +ve amino acids are

A

Arg/Lys

60
Q

at pH 7, -ve amino acids are

A

Asp/Glu

61
Q

Principle of isoelectric focusing:
A _____ gradient is set up in a __________ gel. An ______ is then applied. The protein will move until the ___ = ___, and then stop.

A

pH
polyacrylamide
electric field
pH = pI

62
Q

in IEF, running buffers are required; The anode (__) electrode is connected to a reservoir of _____

A

positive

acid: H3PO4

63
Q

in IEF, running buffers are required; The cathode (__) electrode is connected to a reservoir of _____

A

negative

alkaline: NaOH

64
Q

in IEF, _______ are used to get a high resolution

A

high voltages

65
Q

Applications of IEF include

  • determination of protein isoelectric points ____
  • detection of post translational modification _____
  • detection of single amino acid side chains _____
A

comparison of standard proteins run on the same get (distance vs pH)
protein phosphorylation
variants in serum proteins

66
Q

In 2D electrophoresis, a mixture of proteins can be separated by two biochemical principals; in the first dimension, proteins are separated according to ________. In second dimension, proteins are separated according to _______ using ______.

A

their pIs using IEF
their molecular weights
SDS page

67
Q

non-reducing SDS-PAGE is run without ___

A

DTT

68
Q

optical spectroscopic methods use interactions between ____ and _____ to derive information about the protein. It is dependent on the presence of ____ which can interact with and adsorb light.

A

matter
radiated energy
chromophores

69
Q

what is a chromophore?

A

an atom or group whose presence is responsible for the colour of a compound

70
Q

What is Beer Lambert equation?

A

A=ECL

absorption = absorption coefficient x concentration (M) x path length (cm)

71
Q

what is Beer-Lambert law?

A

absorbance is proportional to the concentrations of the attenuating species in the material sample

Proteins (3 decks)

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