ibt01 lec8 column chromatography - copy

8/11/2019 IBT01 Lec8 Column Chromatography - Copy

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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)


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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


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Cell Disruption Methods

Sonication

French press

Glass bead disruption

Enzymatic lysis(lysozyme)

Detergent lysis

Freeze-thaw

Osmotic lysis(hypotonic solution)

Gentle Method s

Vigoro us Method s


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Remove cell debris by centrifugation

at high speed (~20,000g for 40 mins at 4C)

Pellet

Supernantant (contains protein ofinterest + other biomolecules

Add DNase and RNase to supernatant to

remove contaminating DNA and RNA

Load sample to chromatography column


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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.

Chromatographyis 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.


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Column Chromatography

Most common (and best) approach to purifying largeramounts 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 thestationary 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)


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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)


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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 chargesimmobilized 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


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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


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IEC

Two types

1.Anion exchange chromatography

2.Cation exchange chromatography

ibt01 lec8 column chromatography - copy

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