i'm working on extracting certain fungal enzyme and i need to know how to purify it using ion exchange column chromatography using DEAE-cellulose resin and i have it in powdered form.
If it's purified DEAE-cellulose (white or nearly white in color, such as Whatman DE52), swell it by adding the starting buffer for the purification and stirring gently with a glass rod in a beaker. It's a fragile material. Don't use a magnetic stirrer.
After most of it has settled, pour off the supernatant containing the fine particles. Add more buffer and repeat the process twice more. This is to remove the fine particles that slow down the flow through the column. This resin is too delicate to use with pressure, so chromatography should be carried out under gravity flow, or with a peristaltic pump on the outlet side.
If you have a crude grade, yellow in color, you will have to treat it with acid and base to clean it until it's white, then wash it thoroughly with water.
Swelling DEAE-cellulose in water followed by washing with NaOH/NaCl activates it for ion exchange chromatography to purify negatively charged enzymes, which are then eluted with a salt gradient.
The following are the steps for swelling and activation of DEAE-cellulose, suitable for enzyme purification:
1. DEAE-cellulose swelling (swelling) treatment
2. Alkali treatment (activation)
3. Acid treatment (neutralization and regeneration)
4. Equilibration buffer treatment
- Badges
- Science topic
- Similar topics
- Computer Communications (Networks)
- Simulators