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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
Give an example of a signalling protein
insulin
26
What is the purpose of a binding protein
binds ligands and releases them at target locations
27
Give an example of a motor protein
actin, dynein, myosin
28
Determination of purity
SDS page, isoelectric focusing, peptide mapping
29
Chemical and mass characterisation
N-terminal sequencing, acid analysis, mass spec, SDS page
30
Conformational analysis
Circular dictorism, fluorescence spectroscopy, NMR, X-ray diffraction
31
Analysis of quaternary structure
gel filtration, native gel electrophoresis, analytical centrifugation, light scattering NMR, X-ray differaction
32
SDS Page can be used for
Determination of purity, chemical and mass characterisation
33
NMR can be used for
conformational analysis, analysis of quaternary structure
34
X-ray diffraction can be used for
conformational analysis, analysis of quaternary structure
35
What are the two broad categories of column chromatography?
Non-adsorption and adsorption
36
outline what is meant by non-adsorption in column chromatography
``` gel filtration (size exclusion) proteins distributed between the two phases, travelling at different speeds long column required ```
37
outline what occurs in adsorption column chromatography
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
In SEC, what is a gel matrix?
cross linked polymers with pores acting as molecular sieves. Stationary phase
39
Name 3 cation exchangers
sulphonopropyl methyl sulphonate carbonyl methyl
40
name 3 anion exchangers
quaternary ammonia diethylaminoethyl quaternary aminoethyl
41
ion exchange chromatography separates proteins according to their ability to ______
adsorb the matrix based on their net surface charge
42
Name the 4 steps for IEC
equilibration sample application and wash selective elution regeneration
43
Isoelectric point (pI)
the pH when the net charge of the molecule is zero
44
Retention volume (Vr) =
flow rate (F) x retention time (tR)
45
how do you calculate electrophoresis velocity
velocity = electric force / friction force
46
Polyacrylamide gel electrophoresis (PAGE) separates proteins according to ______
size
47
SDS PAGE separates proteins according to ______
molecular weight
48
Why do you heat up protein prior to SDS PAGE
to denature the protein; smaller proteins move further through the gel
49
What are the components for SDS PAGE?
SDS DTT Glycerol Bromophenol blue
50
what is the function of SDS in SDS PAGE?
it's an ionic and hydrophobic detergent which denatures proteins. It induces a negative charge. All polypeptide chains have similar conformations
51
what is the function of DTT in SDS PAGE?
it reduces disulphide bonds further denatures protein by overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure
52
what is the function of glycerol in SDS PAGE?
increases density - makes bonds sharper
53
what is the function of bromophenol blue in SDS PAGE
dye front
54
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.
low large low charge ``` high small high charge faster ```
55
what is the most common dye for detection in SDS PAGE?
coomassie brilliant blue - binds non-specifically to all proteins, can be used qualitatively to determine concentration
56
what is the most sensitive method of detection in SDS PAGE?
Silver stain - | It's based on the reduction of Ag+ --> Ag
57
What is the most specific method of detection for SDS PAGE?
Western blot - highly sensitive + specific. | Transfers proteins onto membrane. Sensitivity depends on specific antibody. Mw marker is used.
58
Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their __________ along a continuous _____ gradient.
isoelectric point | pH
59
at pH 7, +ve amino acids are
Arg/Lys
60
at pH 7, -ve amino acids are
Asp/Glu
61
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.
pH polyacrylamide electric field pH = pI
62
in IEF, running buffers are required; The anode (__) electrode is connected to a reservoir of _____
positive | acid: H3PO4
63
in IEF, running buffers are required; The cathode (__) electrode is connected to a reservoir of _____
negative | alkaline: NaOH
64
in IEF, _______ are used to get a high resolution
high voltages
65
Applications of IEF include - determination of protein isoelectric points ____ - detection of post translational modification _____ - detection of single amino acid side chains _____
comparison of standard proteins run on the same get (distance vs pH) protein phosphorylation variants in serum proteins
66
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 ______.
their pIs using IEF their molecular weights SDS page
67
non-reducing SDS-PAGE is run without ___
DTT
68
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.
matter radiated energy chromophores
69
what is a chromophore?
an atom or group whose presence is responsible for the colour of a compound
70
What is Beer Lambert equation?
A=ECL absorption = absorption coefficient x concentration (M) x path length (cm)
71
what is Beer-Lambert law?
absorbance is proportional to the concentrations of the attenuating species in the material sample

Proteins (3 decks)

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