I am under the impression that DEAE-cellulose is no longer sold. You can substitute another ion-exchange resin such as QAE-Sephadex if you are planning open-top column chromatography, or QAE-Sepharose if you are planning to use an FPLC. Ion exchange chromatography is usually started at a low ionic strength, at which the proteins bind to the column. Then the column is eluted with a gradient of increasing ionic strength. Fractions are collected during the gradient elution. If the equipment is available, an automated FPLC system is great. However, simpler technologies can also be used.

Dear Raju,

Please read the following publication which covers the answer to your question.

Guide to Ion-Exchange Chromatography

https://www.harvardapparatus.com/downloads/Ion%20Exchange%20Chroma%20SpinColumn%20Guide.pdf

Table of Contents

Introduction ........................................................................2-4

Protocol Samples ....................................................................5

Factors Effecting Selectivity ..................................................6-7

Components of Ion Exchange Media ..................................8-9

Experimental Conditions & Method Optimization..........10-11

References..............................................................................12

Ordering Information............................................................13

Contact Information...........14

Protocol Samples

The SpinColumns are supplied dry and need to be rehydrated, the bed of ionexchange

resin with starting buffer allow 10-15 minutes for rehydration. After

rehydration add a 2ml collection tube to the bottom of the SpinColumn and

centrifuge for 1 minutes at 1000rpm. After centrifugation the protein mixture is

applied. Proteins which are oppositely charged to the media at the starting pH will

bind to it, so displacing the counter ions. Proteins with the same charge as the

resin or with no net charge will not bind and flow straight through the column.

The different proteins bound to the column, one of which will be the protein of

interest, will have different affinities for the ion exchanger due to differences in

their net charge. These affinities can be altered by varying the ionic strength of the

column buffer or alternating the pH of the elution buffer.

Example #1

A mixture of proteins are bound to the anion exchanger. At low ionic strength,

competition between the buffer ions and proteins for charged groups on the ion

exchanger is minimal and so the proteins bind strongly. By increasing the ionic

strength, you increases the competition between the proteins are bound and so

reduces the interaction between the ion exchanger and proteins, causing the

proteins to elute. One can elute the bound proteins by increasing ionic strength

irrespective of whether an anion or cation exchanger was used.

This is usually accomplished by incorporating a linear concentration gradient of

NaCl in the column buffer while keeping the pH constant.

Example #2

A mixture of proteins are bound to the anion exchanger, DEAE-cellulose. As the

pH is lowered, -COO¯ groups on the protein begin to become protonated and so

lose their charge.

As the overall negative charge of the protein decreases its affinity for the resin decreases.

Different proteins will elute from the resin at different pH values as their number

of negative charges decreases to a critical value. Therefore one can resolve the

proteins bound to the column by slowly reducing the pH using a buffer pH

gradient and collecting fractions, each of which will contain different proteins

eluted at different pH values. Conversely, when using a cation exchanger, the pH

gradient would be arranged to increase to elute the bound proteins.

By using either a continuous salt (ionic strength) gradient or a continuous pH gradient

will result in a high degree of protein fractionation based on protein charge

Hoping this will be helpful,

Rafik

I am under the impression that DEAE-cellulose is no longer sold. You can substitute another ion-exchange resin such as QAE-Sephadex if you are planning open-top column chromatography, or QAE-Sepharose if you are planning to use an FPLC. Ion exchange chromatography is usually started at a low ionic strength, at which the proteins bind to the column. Then the column is eluted with a gradient of increasing ionic strength. Fractions are collected during the gradient elution. If the equipment is available, an automated FPLC system is great. However, simpler technologies can also be used.

Thank you very much Adam. In our laboratory, we don't have automatic FPLC. Is it possible to use QAE-Sephadex column without requiring any other instrument?  

 Hi

Before setting up  the column try to screen the ion exchange resins for best binding purpose. Screening can be done using less resin quantity and also gives u an idea about elution profile as which resin binding is weak or strong to proceed for column chromatography runs. As Dr. Adam has mentioned, FPLC is automated technology which saves time and gives best fold purity. If u do not have FPLC unit let me know will guide for gravity separations as well.

Good luck

Dr Mugdha,

I don't have FPLC. How can I proceed? Could you please suggest me.

If you have a small peristaltic pump, a magnetic stirrer, a 2-chamber gradient former, and some appropriately sized flexible tubing, you can do ion-exchange chromatography without an FPLC.

If you don't have a peristaltic pump, you can use gravity to elute the column.

If you don't have a gradient former, you can use 2 beakers connected with tubing. You can also avoid making a continuous gradient and instead use a series of small step elutions of gradually increasing salt concentration.

An automatic fraction collector is nice, but if you don't have that you can collect fractions by hand.

Thank you Dr Adam B Shapiro.

Thank you Dr Adam B Shapiro.

DEAE Cellulose still sold through Santa Cruz

https://www.scbt.com/scbt/product/deae-cellulose-9013-34-7

Just to answer your question DEAE Cellulose is also available through the following Company:

http://srlchem.com/search/search_results/productName/deae%20cellulose