DEAE stands for diethyl aminoethyl (CH3-CH2)2-HN+-CH2CH2-O- , a weak anion exchanger. It needs no activation, only a pH low enough that the amino group is protonated. You should read https://research.fhcrc.org/content/dam/stripe/hahn/methods/biochem/Ion_Exchange_Chromatography_Handbook.pdf.
As Engelbert told you do not need any specific activation.
Generally the new resin are stored in 20% of ethanol. So just wash it with some MilliQ water 3-5 CV (CV = coloumn volumes) to remove the ethanol and than equilibrate it with your binding buffer that have to be selected on the basis of the pI of the protein (on at least 2 unit higher than pi to ensure that the protein is negativelly charged and can bind the coloumn) and at the same time To ensure that the resin is protonated and positively charged, the chromatography should be performed at least 2 pH units below the pKa of the amine group, 10 so generally se use it around ph7-8.
good luck
Manuele
I would not use DEAE ion exchanger with cellulose matrix. Get a more modern DEAE-resin, e.g., with agarose matrix.
Cellulose contracts at higher ionic strength, forming a large "mixing chamber" below the top adaptor of the column. This strongly affects the resolution of the chromatography.
Thank you so much. I appreciate your information. Can we boil DEAE Cellulose for 15-20 min. if we use it for first time?
Cellulose-based resins are very delicate and can easily break apart into small fragments called "fines" that should be removed before pouring the column, otherwise the column clogs and the flow rate is very slow. If you boil the cellulose, it will probably cause a lot of fines to be formed. I would not recommend it.
I presume you mean to know about the preparation of the resin (soaking and swelling). It is recommended that you soak the gel using the chosen equilibrating buffer for 1-3 days. Sometimes, boiling can help reduce the swelling time to a few hours but care must be taking to avoid clogging. In all, follow strictly the manufacturers procedure for the gel preparation. This will be specified by the supplier in the handbill following the resin pack.
I guess the first question I have is why are you using DEAE Cellulose, when DEAE Sephacel, DEAE Sepharose or even a Toyopearl DEAE 650M will work just as well, For these other resins they come pre-swollent nd defining of the resin in not required. Then regarding the regeneration protocol of the Manufacturer, do you know if you want the H+ or the OH- form for the DEAE ? For example to bring your DEAE into the H+ form you need to do a NaOH regeneration step, Water wash and then follow that with a HCl regeneration step, followed by the Water wash and equilibration to your buffer. For the OH- form you reverse the order of NaOH and HCL. Any further questions you may have, please let me know.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column