I am working to purify enzyme using chromatography. Our lab does not have sephadex or sepharose. some papers have used CM cellulose for purification of the enzyme of our interest. But most of the protocols for CM cellulose column preparation is given for ion exchange . Can anyone suggest how to prepare CM Cellulose for gel filtration chromatography ?
Hi there,
CM cellulose is indeed a cation exchanger as it bears carboxylic function. For this purpose working pH has to be above the pK of the carboxylic function (pK is around 4). If you want to use this matrix for gel filtration you should neutralize the negative charge of carboxylic function by lowering the working pH under 3 which might be deleterious to the protein you are interested in. The other solution would be to esterify the carboxylic function to neutralize it... I think the best for you is to buy an appropriate matrix for gel filtration purpose.
Just run the experiment under high salt concentration, 1 M, and this will prevent ion exchange from happening. Of course you need the appropriate matrix to succeed in your separation.
You should obtain some gel filtration resin, rather than trying to use CM-cellulose. Gel filtration resins have a special structure that allows them to perform this function. Cellulose resins do not possess this type of structure.
Dear
Please check :
-DEAE and CM Bio-Gel®A IonExchange Gels Instruction Manual.
-Preparation of carboxymethyl-cellulose gels and their use for immobilization of amyloglucosidase (E.C.3.2.1.3) J. Kučera and J. Hanus.
Also you can read others approaches.
Hoping to helpful.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT310.pdf
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf
https://www.harvardapparatus.com/media/harvard/pdf/Ion%20Exchange%20Chroma%20SpinColumn%20Guide.pdf
http://cccc.uochb.cas.cz/40/8/2536/
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