L8-9: Purification of proteins

Question 1

What is heterologous expression?

Answer 1

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

Question 2

What are the biophysical properties of proteins?

Answer 2

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

Question 3

What are the 2 main methods of separating proteins?

Answer 3

Chromatography
Gel electrophoresis

Question 4

How are proteins isolated?

Answer 4

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

Question 5

What is centrifugal force?

Answer 5

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

Question 6

How can centrifugal force be expressed?

Answer 6

As relative centrifugal force:
RCF=centrifugal force/g

Question 7

What are the 2 forces acting on a centrifuge?

Answer 7

Buoyant force and centrifugal force

Question 8

What does buoyant force depend on?

Answer 8

The viscosity of liquid and density/size of particle

Question 9

What type of centrifugation is needed for organelles and proteins?

Answer 9

Organelles - higher speeds
Proteins - ultra centrifugation

Question 10

Which intrinsic biophysical properties have to be exploited after centrifugation?

Answer 10

Size
Mass
Shape
Interaction
Charge
Hydrophobicity

Question 11

What are the principles of chromatography?

Answer 11

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

Question 12

What are the steps of column chromatography?

Answer 12

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

Question 13

What are the 2 different types of chromatography used?

Answer 13

Thin layer chromatography and column chromatography

Question 14

How can the proteins be detected from the chromatography?

Answer 14

Using other biophysical properties

Question 15

How can protein concentration be calculated in a chromatogram?

Answer 15

By integrating under the peak curve

Question 16

What physical properties can proteins be separated by?

Answer 16

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

Question 17

How are proteins separated based on size exclusion?

Answer 17

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

Question 18

How are proteins separated based on ion exchange?

Answer 18

Based on charge overall, charge distribution and charge density

Question 19

What is the process of ion exchange?

Answer 19

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

Question 20

Name examples of anion and cation exchangers

Answer 20

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

Question 21

How are proteins separated based on hydrophobicity?

Answer 21

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)

Question 22

What is the most commonly used salt for hydrophobicity?

Answer 22

(NH4)2SO4

Question 23

How are proteins separated based affinity?

Answer 23

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

Question 24

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

Answer 24

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

Question 25

What is IMAC? (chromatography)

Answer 25

Immobilised metal affinity chromatography

Question 26

What is IMAC? (chromatography)

Answer 26

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

Question 27

What is electrophoresis?

Answer 27

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

Question 28

What can electrophoresis separate?

Answer 28

Proteins, nucleic acids and glycans

Question 29

What is gel electrophoresis?

Answer 29

When polymer gels act as molecular sieves

Question 30

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

Answer 30

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

Question 31

What are the factors that effect gel electrophoresis?

Answer 31

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

Question 32

What are PAGE variants?

Answer 32

Polyacrylamide gel electrophoresis

Question 33

What is native PAGE? (electrophoresis)

Answer 33

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

Question 34

What is SDS-PAGE?

Answer 34

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Question 35

What are the properties of SDS-PAGE?

Answer 35

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

Question 36

What proteins recalcitrant to denaturation?

Answer 36

Disulfides - require reducing agent Thermostable proteins

Question 37

What are the 2 discontinuous gels?

Answer 37

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

Question 38

What are the buffer components in SDS-PAGE?

Answer 38

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

Question 39

What do gradient gels do in SDS-PAGE?

Answer 39

Increase concentration of acrylamide down gel

Question 40

What buffers are used in agarose gel electrophoresis?

Answer 40

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

Question 41

What is the difference between agarose gel electrophoresis and PAGE?

Answer 41

Resolution isnt as high in agarose

Question 42

How are gels visualised?

Answer 42

Using dyes

Question 43

What are the properties of visualising gels?

Answer 43

They have different sensitivities and different visualisation conditions

Question 44

How can molecular weight be estimated from gels?

Answer 44

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

Question 45

What is the calculation to find molecular weight using gels?

Answer 45

-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

Question 46

What are common problems with gels? (anomalous gels)

Answer 46

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

Question 47

What is absorption spectroscopy?

Answer 47

When a particular wavelength of light is absorbed

Question 48

How is absorption spectroscopy affected by environment?

Answer 48

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

Question 49

What are applications of UV/Vis absorption spectroscopy

Answer 49

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

Question 50

What is the Beer-Lambert Bouger Law

Answer 50

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

Question 51

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

Answer 51

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

Question 52

What are indirect measurements of quantifying proteins?

Answer 52

Lowry Bradford Biuret BCA assay