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Lab Report: DEAE cellulose is an anion exchanger that is positively charged due do a protonated amine group and will attract negatively charged particles. Since charge is highly dependent on pH, the chromatography performed well below the pK of the DEAE group, 10, to ensure the amine is protonated and positively charged. The experiment is performed at a pH of 7.4. At this pH the alkaline phosphatase has a net negative charge and the amines of DEAE are positively charge. The two groups are attracted to each other while the other proteins present in the solution elute out. In order to release the bound AP from the beads, high contractions of NaCl, salt ions, are added to the column to complete with the protein. Upon addition of the salt, the protein is released and eluted and collected in test tube fractions.
From Wikipedia Page: DEAE-C is a weak anion exchanger. Like all anion exchangers, the resin carries a positive charge that interacts favorably with negative charges. The positive charge of DEAE cellulose is due to a protonated amine group. To ensure that the resin is protonated and positively charge, the chromatography should be preformed at least 2 pH units below the pKa of the amine group, 10. [1] The strength of bond between the resin and protein is highly dependent on the pH range in the column and the pI of the protein of interest. The resin is a weak exchanger because it is only partially ionized over most pH values, and an efficient separation with DEAE-C chromatography requires a specific, narrow pH range. Elution of a protein of interest is accomplished by increasing the salt concentration in the column or any means of adding ions that compete for binding to the tertiary amine. NaCl or KCl are typically used because the chloride anions will attach to the resin and displace the protein, allowing the protein to proceed through the column. Adding a suitable buffer may bring the pH close to or below the pI, which causes the protein to lose its positive charges that interact with the resin, thus eluting the molecule from the column.
Cellulose, dextran, agarose and other insoluble complexes are unaffected because they compose inert matrices, hence why are they so often derivatized with strong and weak cation and anion exchangers in chromatography. DEAE-C beads have diethylaminoethyl chains covalently bound to oxygen atoms on the D-glucose subunits of cellulose.
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- ^ Ninfa, Alexander J.; Ballou, David P. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology - 2nd edition. John Wiley & Sons, Inc. ISBN 978-0470087664.