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Structural Biology > Protein Purification > Flashcards

Protein Purification Flashcards

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1
Q

Protein Surface Properties

A
  • heterogenous surface charge and hydrophobicity
  • differing pIs
  • hydrophobic patches
  • ligand binding sites
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2
Q

Aims of Purification

A
  • study enzyme kinetics
  • study enzyme regulation/inhibition
  • study complexes
  • structural analysis
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3
Q

Salting Out

A
  • uses differing surface hydrophobic properties for crude separation
  • ordering of water around hydrophobic patches is unfavorable entropically
  • salting out encourages hydrophobic patches to come together and precipitate
  • releases water so is favorable entropically
  • use divalent ions
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4
Q

Adsorption/Desorption Chromatography

A
  • solid-liquid phases dependent on thermodynamics of interaction of soluble proteins with solid phase
  • interactions with solid phase depends on solution conditions and protein surface heterogeneity/properties
  • design surface particles to interact with the protein
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5
Q

Permeation Chromatography

A
  • liquid-liquid phase method as dependent on the rate of diffusion (kinetics) between liquid phases
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6
Q

Chromatography Considerations

A
  1. selectivity of stationary/separation phase
  2. non specific interactions
  3. hydrodynamics
  4. column design
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7
Q

Langmuir Isotherm

A
  • describes equilibrium of soluble proteins binding to a solid phase
  • see derivation in notes *
  • implies good adsorption is dependent on high binding site concentration
  • low dissociation constant
  • modulate and reduce binding to elute protein
  • elution when alpha (fraction of bound protein) is 0.5 or less
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8
Q

Adsorption Considerations (Isotherm)

A
  • avidity effects
  • sites not of equal strength
  • Pt not constant in band
  • concentration of sites varies
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9
Q

Dynamic Effects

A
  • as solution flows past solid phase there is a balance between flow and equilibrium rates
  • dispersion: protein band spreading
  • both diffusion and turbulent flow contribute to dispersion
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10
Q

Diffusion

A
  • regions ahead and at back of band have no protein so molecules will diffuse into these regions
  • leads to elution as Gaussian shaped curve with increased band volume
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11
Q

Turbulent Flow

A
  • as solution passes through solid phase it flows through channels between particles of solid phase
  • these are uneven so turbulent of eddy flow ensues
  • fast flow: less broadening but more turbulent flow
  • slow flow: more broadening but less turbulent flow
  • see equation in notes *
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12
Q

Self-Sharpening

A
  • sharpening of band as the back tails
  • middle part moves faster
  • at lower Pt protein is more likely to bind so migration down column slows so the leading edge sharpens and trailing edge extends
  • if a molecule for some reason gets ahead of its band, it enters a zone in which it is more strongly retained, and will then run more slowly until its zone catches up
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13
Q

Ion Exchange Chromatography

A
  • separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one unit away from the isoelectric point of a protein
  • anion or cation exchange chromatography
  • equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column
  • elution done by running anions/cations at high concentration through column
  • fraction of bound sites will titrate with the ratio of charged to non charged groups
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14
Q

Considerations of Ion Exchange

A
  • capacity depends on molecular weight of protein
  • overloading causes fractionation by size
  • pH at exchanger surface may differ from bulk solution
  • ensure buffer species don’t bind preferentially
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15
Q

Band Sharpening

A
  • as salt is increased so does Kd therefore fraction of bound sites decreases and you get elution/increased mobility down column
  • trailing edge catches up with leading edge for improved resolution
  • essentially trailing edge can’t go as fast as it has more bound time so you get salt induced mobility
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16
Q

Chromatofocussing

A
  • Chromatofocusing is a protein-separation technique that allows resolution of single proteins and other ampholytes from a complex mixture according to differences in their isoelectric point.
  • use pH gradient to get protein to rebind to column and allow trailing edge to catch up
  • start above pI for binding, then lower, then raise
  • gradient created using exchanger/buffer with range of pKa values
17
Q

Hydrophobic Interaction

A
  • similar to ion exchange but based on hydrophobic interactions
  • solid matrix carries bound aryl/alkyl groups
  • proteins loaded in high salt to promote salting out effect and eluted with decreasing gradient
18
Q

Reverse Phase Chromatography

A
  • similar to hydrophobic interactions but solid matrix carries more hydrophobic interactions
  • organic solvents
  • denatured proteins
  • using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds
19
Q

Affinity Chromatography

A
  • specific interaction between immobilised ligand and protein
  • must consider protein sticking at high concentrations, lower ligand capacity, and ion exchange effects of ligand
  • can use fusion proteins, lectins, dye, antibodies
  • for elution you compete with a solution ligand or change I or pH
20
Q

Gel Filtration

A
  • liquid liquid phase by restricted diffusion
  • solid phase is used by no interaction with mobile phase
  • need high concentrations
  • porous solid beads: large molecules excluded from the pores and travel in the extraparticular void
21
Q

Selectivity Curve

A
  • shows variation of Kav (fraction of internal volume that is accessible to protein) as a function of log molecular weight
  • for given pore size distribution
22
Q

Protein Electrophoresis

A
  • migration of proteins is an applied electric field

- rate of migration proportional to field strength, ionic strength, net charge, temperature, viscosity

23
Q

Denaturing Gels

A
  • SDS Page
  • denaturated by heating, reducing agent, and SDS detergent
  • SDS binds to protein on constant weight basis
  • protein intrinsic charge is swamped by the charge on the bound SDS thus all proteins have the same charge and negative density so migrate due to size
24
Q

Discontinuous Gels

A
  • gel is divided into two discontinuous parts, resolving and stacking gel, both have different concentrations of polyacrylamide
  • improves resolution by entering the run gel at a higher concentration (limit broadening)
  • in stacking gel a boundary forms between Gly and Chlorine trapping proteins between the two regions and compressed into a narrow bands
  • Glycine has very low net charge at pH 6.8 of stacking gel, so it has low mobility.
  • The proteins are separated according to the principle of isotachophoresis and form stacks in the order of mobility (stacking effect). Mobility depends on net charge, not on the size of the molecule. Proteins move towards anode slowly at constant speed till they reach limit of separation gel. Suddenly, frictional resistance increases but glycine is not affected and it passes the proteins and becomes highly charged in resolving zone. Proteins present in homogeneous buffer start to separate based on principles of zone electrophoresis. Now their mobility depends on size as well as charge
25
Q

Native PAGE

A
  • proteins not denatured
  • migration dependent on size and charge
  • gel have higher pH than protein
  • closer pH to pI increases running time and separation
26
Q

Isoelectric Focusing

A
  • migration through preformed pH gradient across gel
  • gradient formed from ampholytes
  • proteins migrate to pI and lose chargge so stop migration
  • high resolution
27
Q

2D Page

A
  • migration through pH gradient then rotated and migrate through SDS gel
  • incredible resolving power
28
Q

Capillary Electrophoresis

A
  • Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage
  • high efficient with fast separation/small sample size
  • cons: sticky molecules and reproducibility
29
Q

Electrosmosis

A
  • excess cations in the diffuse stern double layer flow towards the cathode exceeding the opposite flow towards the anode
  • net flow occurs as solvated cations drag along solution
30
Q

Capillary Isoelectric Focusing

A
  • allows proteins to be separated by electrophoresis in a pH gradient generated between cathode and anode
  • solutes migrated to point where net charge is lost, stopping migration to focus into tight zone
  • no separation based on size

Structural Biology (10 decks)

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