Separation techniques used for

Protein purification

Column Chromatography Techniques

Ion exchange (cation /anion)
Size exclusion chromatography (also called
gel-filtration chromatography)
Affinity chromatography
HIC (hydrophobic interaction chromatography)


Protein Isolation & Purification
Proteins synthesizes inside cells /
secreted outside the cells in media
Cells (or media) are harvested by
centrifugation using a centrifuge.
Intracellular (inside cell) proteins are harder to purify
Require cell disruption, separation, removal of cell debris, DNA,
RNA, lipid
Extracellular (outside cell) proteins are easier to purify
No cell disruption needed, just isolate
Cell Disruption Methods
Sonication
French press
Glass bead disruption

Enzymatic lysis
(lysozyme)
Detergent lysis
Freeze-thaw
Osmotic lysis
(hypotonic solution)
Gentle Methods
Vigorous Methods
Remove cell debris by centrifugation
at high speed (~20,000g for 40 mins at 4C)

Pellet
Supernantant (contains protein of
interest + other biomolecules
Add DNase and RNase to supernatant to
remove contaminating DNA and RNA
Load sample to chromatography column
Chromatography

Is the collective term for a set of laboratory techniques used for the
separation of molecule from a mixture.

Mobile phase
Stationary phase

It involves passing a mixture dissolved in a "mobile phase" through a
stationary phase, which separates the molecule of interest from other
molecules in the mixture based on differential partitioning between the
mobile and stationary phases.
Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of
which is stationary (stationary phase) while the other (the mobile phase)
moves in a definite direction.
Column Chromatography
Most common (and best) approach to purifying larger
amounts of proteins
Able to achieve the highest level of purity and largest
amount of protein with least amount of effort and the
lowest likelihood of damage to the protein product
Standard method for pharma industry
Column chromatography is a separation technique in which the
stationary bed is within a tube. The particles of the solid stationary
phase or the support coated with a liquid stationary phase may fill the
whole inside volume of the tube (packed column)
Column Chromatography
Column Chromatography
Can be done either at atmospheric pressure
(gravity feed) or at high pressure (HPLC, 500-
2000 psi: column used for HPLC have fine material beads =
better resolution because more interaction sites = but pressure has
to be applied to get the mobile phase flowing)

Four types of chromatography:
Affinity chromatography
Gel filtration (size exclusion) chrom.
Ion exchange chromatography (IEC)
Hydrophobic Interaction chrom. (HIC)
Ion Exchange Chromatography
(IEC)
Principle is to separate on the basis of charge
adsorption
It relies on charge-charge interactions between
the proteins in your sample and the charges
immobilized on the resin
Positively charged proteins are reversibly
adsorbed to immobilized negatively charged
beads/polymers
Negatively charged proteins are reversibly
adsorbed to immobilized positively charged
beads/polymers
Ion Exchange Chromatography
Has highest resolving power
High capacity
Generally preparative
Widespread applicability (almost universal)
Most frequent chromatographic technique
for protein purification
Do not alter protein
Used in ~75% of all purifications
IEC
Two types
1. Anion exchange chromatography
2. Cation exchange chromatography