Protein Purification Flashcards
Why study proteins?
- Structural analysis
- Sequential analysis
- Protein analysis -> final product of a gene expression
- Proteome analysis
- Functional analysis
Basis of protein purification strategies?
- Cell lysis (break cell open)
- Centrifugation (separate cell elements)
- Fractionation
- Separation techniques/detection techniques
- Functional analysis
What are the two types of cell lysis disruption methods?
1) Chemical disruption
2) Physical disruption
Cell Lysis: Chemical Disruption
- Rapid, gentle, efficient, and reproducible method leading to high protein yield
- Disrupts the lipid membrane and/or cell wall
- High [salt] and [detergents] are not compatible with protein assays and mass spectrometry
Cell Lysis: Physical Disruption
- May require expensive equipment
- Reproducibility may vary
- Mechanical methods are generally not compatible with high-throughput and small volumes
Chemical Disruption Methods
1) Osmotic lysis: dilute sucrose solution hypotonic cellular environment = swell and burst of cell
2) Organic solvents: for plants, permeating the cell walls and membranes and disrupt the cell wall
3) Chelating agent: EDTA disrupt gram negative microorganisms, chelates the cations, leaving holes in the cell walls
4) Detergent: disrupt the distinct interface b/w hydrophobic and hydrophilic systems
5) Chaotropic agents: urea and guanidine disrupting the structure of water and making it a less hydrophilic environment, and weakening interactions among solute molecules
Physical disruption methods:
1) Blenders: Mechanical lysis, rotating blades grand and disperse cell and tissues
2) Sonicators: High-frequency sound waves shear cells
3) Liquid Nitrogen freeze-thaw cycles: Repeated cycles of freezing and thawing disrupt cells through ice crystal formation
4) Mortar/pestle: grinding, frozen in liquid nitrogen
5) French Press: cell or tissue suspensions are sheared by forcing them through a narrow space
Fractionation: “Salt in” & “Salt out”
At high [salt], salt ions and protein surface charges compete for water molecules (finite resource)
As H2O molecules available for hydration shells become more and more scarce, the least soluble molecules aggregate and precipitate
Dialysis: get rid of salt or fractionate
Salt precipitation usually followed by dialysis
- Protein solution is placed in a semipermeable dialysis bag and placed in a large volume of buffer
- Smaller molecules (salt ions) can diffuse through the small pores in the dialysis bag and into the buffer until equilibrium is reached
- Several changes of dialysis buffer are required to remove all salt
- Larger molecules (proteins) remain inside the dialysis bag
Separation by Size: Gel Filtration Chromatography
Proteins of interest interact differentially with the phases: stationary and mobile phase
- Carbohydrate polymer bead
- Small molecules enter the aqueous spaces within beads
- Large molecules cannot enter beads
Separation by Charge: Ion Exchange Chromatography
Proteins of interest interact differently with the phases: stationary and mobile phases
- Bead of resin can be +/- charged or neutral depending on the group
- Anion exchange/cation exchange chromatography
- The strength of the adsorption increases with the increased net charge
- Elution by exchangeable counterions, pH change
- Can be a gradient
- Counterion competes with adsorbed protein
Separation by Affinity: Affinity Chromatography
Separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions.
Separation by Electrophoresis: SDS-PAGE
Separated by molecular weight
Smaller molecules travel further in the gel than larger ones.
SDS-PAGE denatures and reduces protein
Direction is from (-) to (+)
Isoelectric Focusing Gel Electrophoresis (IEF)
pI = pH at which net charge on the molecule is zero
Below pI = (+) charge
Above pI = (-) charge
Proteins will move toward the electrode with the opposite charge (during motion, they will lose or gain protons)
Proteins are separated in a pH gradient depending on the isoelectric points
Polyacrylamide is used as a solid surface
2D PAGE
The technique of IEF and SDS PAGE combined
On the base of pI followed by SDS PAGE
