Ion-exchange

Crude enzyme extract

Salt precipitation

Gel filtration Ion exchange

Source of protein

Extraction

Separation

Purity & characterization

Ion-exchange

Topic 4: Ion-Exchange Chromatography

What is the principle behind this method?What are the types of ion-exchange chromatography available? At which protein purification step is this method a target?Procedure and exploitation. Can I use any ion-exchange resins and buffers in together?Think creatively

Source of protein

Extraction

Separation

Purity & characterization

Ion-exchange

Principle of ion exchange chromatography

Proteins are charged molecules. At specific pH, it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage.

In Ion exchange chromatography separation is based on the charges carried by the protein molecules

pH increase

pH =pIcationic anionic

Ion-exchange

Since proteins can have net positive or negative charge, it becomes obvious that two forms of ion-exchanger would be relevant

Negatively (anionic species) or positively (anionic species) charged molecules can bind to functional groups bearing the opposite charges.

Possible to have two types of ion exchangersAnion exchanger (anion exchange chromatography)Cation exchanger (cation exchange chromatography)

Ion-exchange

Anion exchange chromatography

+

++

+ +

Anion exchanger is positively charge

--

-

-

+ -

-

Counter ions are negatively charged

Positively charged functional groups are bound to the insoluble matrix

Ion-exchange

Anion exchange chromatography

+

++

+ +

--

-

-

+ -

-

Negatively charged proteins can replace the counter ions and be bound to the ion exchanger.

Counter ions in the eluting buffer can then exchange for the protein species, thus releasing the proteins from the ion exchanger

Separation is based on the degree of of binding strength of the proteins to the ion exchanger

Ion-exchange

Ion exchangers – Functional groups

+

++

+ +

--

-

-

+ -

-

-

--

- -

++

+

+

-

+

+

Anion exchangerAminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE-)

Counter ions

Cation exchangerCarboxymethyl (CM-) Phospho Sulphopropyl (SP-)

Ion-exchange

Topic 4: Ion-Exchange Chromatography

What is the principle behind this method?What are the types of ion-exchange chromatography available? At which protein purification step is this method a target?Procedure and exploitation. Can I use any ion-exchange resins and buffers in together?Some recent development

Ion-exchange

Ion-exchange chromatography can be used after salt precipitation step or after gel-filtration.

1. Requires dialysis of sample prior to ion-exchange

2. Can be used directly after gel filtration

1

2

Ion-exchange

Topic 4: Ion-Exchange Chromatography

What is the principle behind this method?What are the types of ion-exchange chromatography available? At which protein purification step is this method a target?Procedure and exploitation.Can I use any ion-exchange resins and buffers in together?Some recent development

Ion-exchange

Set up for ion-exchange chromatography

Ion-exchange

Steps involved

2

1

3

How?

Ion-exchange

Rationale behind steps

Steps1. Sample application and

adsorption and equilibration

2. Elution of column with specific buffers to achieve protein separation -

salt gradientionic strength gradient

3. Regeneration of ion-exchangers

RemarksSample diffuses into ion-exchanger surface and matrixUnbound proteins will be removed

Weakly bound proteins will be eluted first, followed by those which are tightly bound

Ion-exchanger can be reused

Ion-exchange

Which type of ion exchanger should I use?

Establish if you need to use an anion or cation exchangerConsider the strength and capacity of ion exchangerDo you have any preference for matrix type?Which buffer should I use with the selected ion exchanger

Ion-exchange

Choosing your ion-exchanger: know your proteins

Stability of proteins stable below pI value, use cation-exchangerstable above pI value, use anion-exchanger

Molecular size of proteins<10,000 mw, use matrix of small pore size10,000-100,000 mw, use Sepharose equivalent grade

Other specific requirementsInactivation of specific buffer types, then limit choice of ion-exchanger

Ion-exchange

Important to consider the stability of proteins in choice of ion-exchangers. Isoelectric focusing can be used to identify suitable ion-exchanger type

pH<pI

pH>pI

pH=pI

Ion-exchange

What is the ideal condition (pH) for substance to bind to ion-exchanger?

Ion-exchange

If too high a pH is chosen, binding of substance to ion-exchanger becomes strong, and elution becomes difficult and high salt concentrations may have to be used

Ion-exchange

Strength and Capacity of Ion-exchangers

Strengthdetermined by functional groupstrong or weak ion-exchangers -reference to extent of ionisation with pHstrong - complete ionisation over a wide pH range

Example:DEAE = weak ion-exchangerSP = strong ion-exchanger

Capacityquantitative measure of ion-exchange ability to bind ionsdepends on number of available functional groupInformation usually provided by manufacturer

Ion-exchange

Ion-exchangers: Matrix available

SephadexSepharoseCellulosePolyacrylic acidPolystyrene

Ion-exchange

Choice of buffers. Use the correct buffers

ConsiderationBuffer types

Cationic buffers with anion-exchangersAnionic buffers with cation-exchangers

pH1 unit + pI value of proteinfacilitate binding

ionic-strengthfor separation

DEAE: Tris, Imidazole, pyridine

CM: acetate, citrate, phosphate

anion exchanger: 1 unit > pIcation exchanger: 1 unit< pI

determined experimentallySalts used: NaCl, KCl

Ion-exchange

Ionic strength of buffer & elution types

To achieve good separation, choice of buffer ionic strength is as important as choice of ion-exchangersInitial strength: LOW ionic strengthDifferent approaches taken for different purposes

isocratic elutionionic strength manipulated to elute or retain protein

gradient elutionincreasing ionic strength (low to high)continuous gradientstep gradient

Ion-exchange

Elution gradient and flow rates are important factors in ion-exchange chromatography

Ion-exchange

Flow rate 8ml/h

Gradient 0 to 0.3M NaCl

Flow rate 8ml/h

Gradient 0 to 0.4M NaCl

Gradient steepness can affect resolution

Ion-exchange

Same elution gradient but different flow rate

Flowrate 8 ml/h

Flowrate 20 ml/h

Ion-exchange

Think creativity

You do not have to bind the protein of interest to the ion-exchanger. It can be reversed.

You can always bind what you do not want to the ion-exchanger. Sometimes this can be an

advantage.

Ion-exchange

Application. Using both anion and cation exchangers

Ion-exchange

Crude enzyme extract

Salt precipitation

Gel filtration Ion exchange

Dialysis step

Concentration step needed

Ion-exchange

Summary

Know what is anion and cation exchange chromatographyKnow the availability of different types of ion-exchange chromatography resins and how to apply themAble to decide at which stage of protein isolation should we use this methodAble to follow published procedure involving this procedure