Hello, I have been using DEAE Sepharose Fast Flow to purify carbohydrates many times and it seems to lose its binding capacity. I washed the resin with30% isopropanol, water, 5% acetic acid, water, 0.2 M NaOH, water, and recharge with T1000 buffer (50 mM Tris pH 8.0 and 1 M NaCl). The resin still doesn't bind to my materials that well. Is acetic acid treatment bad for the resin? And if it's bad, is there a way to reactivate the resin at all? Thank you very much.
Article Characterization of the chemical and functional stability of...
I think DEAE comes as the protonated form? If so, it looks like your protocol my leave it in the chloride form. IIRC, chloride displaces protons and your carbohydrate may not be able to easily displace chloride. Try washing copiously with an acid as the last step; the law of mass action converts the column back to the protonated form.
Also, I thought I saw a limit on acetic acid concentration but couldn't find it quickly. Please check the documents here: http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17070901
Is irreversible binding something to consider here (could it be your ligand's releasing incompletely)? It might be advantageous to consider other options besides DEAE, at least to save yourself some time.
It may need ion additions by using salts at a slight alkaline pH in cell free extract. Activation of plant quinate:NAD' 3-oxidoreductase by Ca2+and calmodulin.The cell-free extract, that served as the source of the NPC also showed a reactivating and protective effect in different pro- and eukaryotic organisms inactivated by different stress factors. You are following right method with30% isopropanol, water, 5% acetic acid, water, 0.2 M NaOH, water, and recharge with T1000 buffer (50 mM Tris pH 8.0 and 1 M NaCl). But preincubation with CFE then incubation with ethanol may also reactivate inactivated DEAE Sepharose.
Hi Jack Silver, Anthony Burgess-Cassier, and Ravi Kant Upadhyay
First of all, thank you very much for trying to help me. I got Jack's comment 8 days ago and I have tried his idea first. I didn't have a chance to respond until now. I think the idea about the resin gets deprotonated might be the case. I washed my resin with 5% acetic acid and then recharge with the high salt. It seemed that my resin could bind to carbohydrate once again. So this answered my question that the acetic acid did not ruin the resin. But the question of whether it works as well as before is still unclear to me. When I use it next time, I think I'll just wash the resin with NaOH first, then the acetic acid before recharging the resin.
Thanks Anthony and Ravi anyway for trying to help. I am greatly appreciate your suggestions.
Regards,
Decha S
- Badges
- Science topic
- Similar topics
- Entomology
- Resistance