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Biochemistry > L8-9: Purification of proteins > Flashcards

L8-9: Purification of proteins Flashcards

1
Q

What is heterologous expression?

A

The use of bacteria/yeast/mammalian/insect cells to make protein of interest which allows lots of it to be obtained but it isnt always in its native state

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2
Q

What are the biophysical properties of proteins?

A

Size
Mass
Shape/structure (primary to quaternary & folding)
Interactions
Redox potential
Charge
Hydrophobicity
Electromagnetic properties

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3
Q

What are the 2 main methods of separating proteins?

A

Chromatography
Gel electrophoresis

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4
Q

How are proteins isolated?

A

Using centrifugation :
Cells are homogenised then blended then filtered and centrifuged

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5
Q

What is centrifugal force?

A

A force appearing to act on an object when viewed in a rotating frame of reference

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6
Q

How can centrifugal force be expressed?

A

As relative centrifugal force:
RCF=centrifugal force/g

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7
Q

What are the 2 forces acting on a centrifuge?

A

Buoyant force and centrifugal force

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8
Q

What does buoyant force depend on?

A

The viscosity of liquid and density/size of particle

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9
Q

What type of centrifugation is needed for organelles and proteins?

A

Organelles - higher speeds
Proteins - ultra centrifugation

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10
Q

Which intrinsic biophysical properties have to be exploited after centrifugation?

A

Size
Mass
Shape
Interaction
Charge
Hydrophobicity

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11
Q

What are the principles of chromatography?

A

Sample of interest (proteins) have differential interaction strength between mobile and stationary phase

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12
Q

What are the steps of column chromatography?

A

1- protein mix in buffer added to column with stationary phase and suitable buffer
2- Buffer used as mobile phase and flows through column, proteins interacting with stationary phase move much slower than ones remaining in the mobile phase
3- Proteins in mobile phase exit the column first

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13
Q

What are the 2 different types of chromatography used?

A

Thin layer chromatography and column chromatography

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14
Q

How can the proteins be detected from the chromatography?

A

Using other biophysical properties

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15
Q

How can protein concentration be calculated in a chromatogram?

A

By integrating under the peak curve

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16
Q

What physical properties can proteins be separated by?

A

Affinity, Ion exchange, size exclusion, hydrophobic interaction, reversed phase and multimodal chromatography

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17
Q

How are proteins separated based on size exclusion?

A

Gel filtration, separates based on size (hydrodynamic radius), large separates first

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18
Q

How are proteins separated based on ion exchange?

A

Based on charge overall, charge distribution and charge density

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19
Q

What is the process of ion exchange?

A

Mix of proteins in buffer loaded into cation exchanger
+ive charged proteins adsorb to media displace Na+
Pulse of buffer is formed
pH decreases to aviod drastic ionic strength

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20
Q

Name examples of anion and cation exchangers

A

Anion:
Quaternary ammonium
Diethylaminoethyl
Diethylaminopropyl
Cation:
Sulfopropyl
Methyl sulfonate
Carboxymethyl

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21
Q

How are proteins separated based on hydrophobicity?

A

Differences in surface hydrophobicity, media modified with hydrophobic alkyl (-CH3)/aryl (-phenyl) groups, high salt concentration favours interaction of protein with media
(works like size exclusion chromatography)

22
Q

What is the most commonly used salt for hydrophobicity?

A

(NH4)2SO4

23
Q

How are proteins separated based affinity?

A

Stationary phase functionalised with chemical/protein that binds protein of interest

24
Q

Name examples of chemicals that bind proteins of interest in affinity chromatography

A

ProteinA- IgG
Streptactin- Biotin or Strep-tag
NTA (nitriloacetic acid)- metals His-tag
Heparin- DNA binding proteins

25
Q

What is IMAC? (chromatography)

A

Immobilised metal affinity chromatography

26
Q

What is IMAC? (chromatography)

A

Required recombinant protein expression, His-tag (6-10 histidine residues added to protein), at N- or C- terminus, Histidine binds to metals like nickel and cobalt, produces pure protein quick

27
Q

What is electrophoresis?

A

The movement of dispersed particles relative to a fluid under the influence of a spatially uniform electric field

28
Q

What can electrophoresis separate?

A

Proteins, nucleic acids and glycans

29
Q

What is gel electrophoresis?

A

When polymer gels act as molecular sieves

30
Q

What are 2 different types of gels and what are they made of?

A

Agarose - linear polymer (polysaccharide)
Acrylamide - Organic compound with multiple functional groups

31
Q

What are the factors that effect gel electrophoresis?

A

-Net charge of molecule
-Size of molecule
-Electric field strength (can denature)
-Properties of gel
-Properties of running buffer (pH, counter ions, Salts)
-Temperature

32
Q

What are PAGE variants?

A

Polyacrylamide gel electrophoresis

33
Q

What is native PAGE? (electrophoresis)

A

Separation of acidic proteins
Separation by charge/size
Samples added to buffer with dye/glycine/buffer
Good for looking at protein complexes
Post-translational modifications

34
Q

What is SDS-PAGE?

A

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

35
Q

What are the properties of SDS-PAGE?

A

Protein denatured with heat/SDS
SDS in gel/buffer: laemmli buffer
Formation of mixed proteins: SDS micelles
Charge by protein masked
Separation by mass

36
Q

What proteins recalcitrant to denaturation?

A

Disulfides - require reducing agent
Thermostable proteins

37
Q

What are the 2 discontinuous gels?

A

pH 6.5 stacking gel (focuses proteins before separation)
pH 8.8 resolving gel (separates proteins)

38
Q

What are the buffer components in SDS-PAGE?

A

Glycine/chloride counter ions (migration)
SDS (denaturation)
Tris (pH 8.3)

39
Q

What do gradient gels do in SDS-PAGE?

A

Increase concentration of acrylamide down gel

40
Q

What buffers are used in agarose gel electrophoresis?

A

Tris pH 8.3
Acetate/Borate (counter ion)
EDTA (chelating agent)

41
Q

What is the difference between agarose gel electrophoresis and PAGE?

A

Resolution isnt as high in agarose

42
Q

How are gels visualised?

A

Using dyes

43
Q

What are the properties of visualising gels?

A

They have different sensitivities and different visualisation conditions

44
Q

How can molecular weight be estimated from gels?

A

-comparing migration of samples vs standard curve
-protein/nuc acid standards/markers run on gels

45
Q

What is the calculation to find molecular weight using gels?

A

-measure migration dist of standards and dye front from top
-calculate relative motility standards (Rf)
-measure for unknowns
-plot log(MW) of standards vs Rf
-plot best fit and calculate eqn
-solve

46
Q

What are common problems with gels? (anomalous gels)

A

Smileys (high voltage)
Melting gels (too hot)
Smearing (overloading)
Contaminants (double dipping)
Bubbles
Speckles
Floaty samples
Leaky gel tanks

47
Q

What is absorption spectroscopy?

A

When a particular wavelength of light is absorbed

48
Q

How is absorption spectroscopy affected by environment?

A

Bathochromic- shift to longer wavelength
Hypsochromic- shift to shorter wavelength

49
Q

What are applications of UV/Vis absorption spectroscopy

A

Quantification of protein/nuc acids
Protein unfolding
Nuc acid unfolding
Characterisation of substrate/cofactor binding proteins
Biochemical assays

50
Q

What is the Beer-Lambert Bouger Law

A

𝐴= 𝜀𝓁𝒸
𝜀- molar attenuation coefficient
𝓁- optical path length
𝒸- concentration of attenuating species

51
Q

What are the key assumptions of the Beer-Lambert-Bouguer law?

A

Monochromatic/parallel illumination
Homogeneous solution
No scatter from medium
Linear absorbance change with conc
Linear response of detector

52
Q

What are indirect measurements of quantifying proteins?

A

Lowry
Bradford
Biuret
BCA assay

Biochemistry (10 decks)

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