Protein purification & Electrophoresis

Question 1

(1) Salting Out

Answer 1

• protein purification method
• exploiting different surface hydrophobic properties and proteins for crude separation
• hydrophobic patches are not solvated by water molecules –> ordering of water clathrates around hydrophobic surface (hydrophobic effect) and cause increase of order in system (entropic penalty)
• initially, patches can be exposed but if salt is added, salt will preferentially solvate in bulk water
• this creates more partition between ordered water & bulk water –> results in higher ordering of system (more entropically unfavourable)
• salt added & reaches certain conc where entropic penatly is too unfavourable –> hydrophobic patches will come together in attempt to release ordered water and reduce entropy
• if enough surfaces come together, the protein will precipitate, & can centrifuge the solution to collect it
• protein w/higher surface hydrophobicity will precipitate first so can perform experiment w/increasing salt concentrations to separate diff proteins
• works best w/divalent anions e.g. sulphate (so4 2-); most effective salt is ammonium sulphate

ranking:
anions: sulphate > hydrogen phosphate > acetate > Cl- > No-3
cations: NH+4 > Na+ ~ K+ > Li + > Mg2+

Question 2

(2) Families of chromatography

Answer 2

1. adsorption/desorption (most efficient)
   -solid phase interacts w/protein (bind)
   -important to understand thermodynamics of proteins interactions w/solid phase
2. permeation
   -no interaction between protein & solid phase (only to impart filtering); only liquid-liquid phase method
   -only depend on rate of diffusion of molecules between liquid phases (depends on size & shape)

Question 3

(2) Set up and considerations in chromatography

Answer 3

set up: need glass column w/means of flowing proteins/buffer through solid phase (eg. gravity/pump)

considerations:
1. selectivity of stationary phase (use what to bind to which protein)
2. surface chemistry (don’t want non-specific interaction)
3. hydrodynamics (how fast flow should be; to prevent turbulence)
4. column design (what size/how packed)

Question 4

(2) Adsorption/Desorption chromatography (general)

Answer 4

• need to engineer protein surface chemistry & solution conditions to induce binding of protein to solid phase (e.g. engineer -ve charge for protein to bind to +ve charge solid phase)
• adsorption/desorption exists in equilibrium which can be explained by Langmuir Isotherm

Question 5

(2) Langmuir Isotherm

Answer 5

θ = [S] / Kd + [S]

θ = fraction of surface sites occupied (0-1)
[S] = concentration of protein in solution; hard parameter as changing all the time as protein binds and leaves solid phase
Kd = dissociation constant for protein surface complex

• process of collision for interactions to happen is random; rate is limited to the random collisions which is broadly similar across proteins (only vary in some cases e.g. conformational change must happen before fully binding)

θ = q/ Γ

q = concentration of binding sites occupied
Γ = tot. conc of binding sites

[S] = Pt -q (total prot. conc - sites occupied)

But more interested in α (the fraction of protein bound) than θ (amount of sites occupied -is created to exist in excess to prevent limitations of binding)

α = q/Pt
α = Γ/Kd + Γ

• as solution conditions change (pH, pI, polarity, so does Kd, hence α
• Γ is physically activated w/column, depends on type bought so can’t be changed
• α only depends on Kd & can manipulate Kd for: adsorption (α 0.8-1) or desoprtion/elute (α 0.5 or less)
• still have assumptions:
  1. adsoprtion sites are not of equal strength (range of Kd’s)
  2. Pt isnt constant due to diluting
  3. avidity effects (large proteins can contact column in multiple points -not considered in equation)
  4. concentration of binding sites = not homogenous across surface

Question 6

(2) Dynamic effects of adsorption/desorption

Answer 6

• ads/des not instantaneous; proteins need a certain amount of time in contact w/solid phase to reach equilibrium

if flow too fast = causes turbulent flow
* vortex and eddy currents occur when liquid flows through uneven channels between particles and solid phase because the solution can flow back up
* this disrupts the continuous flow of solution down the column & will result in froth/mixing effect w/solid phase

if flow too slow = diffusion is occurring
* column is buffered to prevent air in the system drying out solid phase which would result in low resolution so buffer is present in both above & below protein band
* buffer has no protein, so by osmosis, at front and rear of protein band, protein will diffuse from high conc to low conc

• both turbulent flow and diffusion contribute to dispersion (spreading of protein band across the column
  *dispersion causes elution of protein to be in Gausian profile not in blocks
  *need balance between establishing eq & flow rate to minimize dispersion (because high dispersion = low resolution); and also to reduce mixing of different proteins in solution

Question 7

(2) Ion exchange chromatography (IEC)

Question 8

(2) Chromatofocussing

Question 9

(2) Hydrophobic interaction chromatography (HIC)

Question 10

(2) Reverse phase chromatography

Question 11

(2) Affinity chromatography

Question 12

(2) Fusion proteins

Question 13

(2) Gel filtration chromatography (permeation)

Question 14

(3) Protein electrophoresis

Question 15

(3) Denaturing gels (SDS-PAGE)

Question 16

(3) Native gel PAGE

Question 17

(3) Isoelectric focusing (IEF)

Question 18

(3) 2D-PAGE

Question 19

(3) Capillary electrophoresis

Question 20

(3) Capillary isoelectric focusing