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Preparation of Chelexed Milli-Q H₂O

Chelex resin is a styrene divinylbenzene copolymer containing paired iminodiacetate ions which chelate polyvalent metal ions. It is regenerated in dilute acid and operates in solutions of pH 4 or higher. Resin selectivity for different divalent cations varies and depends on a variety of factors, but the selectivity for divalent cations is much higher than for monovalent cations (~5000 to 1).

Equipment/Materials

• Peristaltic PumpPro TPM
• Peristaltic tubing with purple clips
• Tygon tubing (R-3603)
• Chelex 100 resin (Bio-Rad Analytical Grade Chelex Resin 100-200 mesh, sodium form, cat #142-2832)¹

Column preparation and use

1. Slurry the resin in Milli-Q H₂O and pour the column. Allow multiple bed volumes of Milli-Q H₂O to pass through the column so that it is well packed.
2. Wash your collection container with 5 mM EDTA (pH 7) and with the first few thousand milliliters of water collected from the column.
3. Set the peristaltic pump at 30 = 250 ml hr^-1.
4. Run Milli-Q H₂O through column into container.

Note:Since the sodium form of Chelex resin is basic (pH ~11), multiple washes with Milli-Q H₂O are required to approach neutral pH. Filtration can also be used to more rapidly prepare the resin.

Column Regeneration

1. Wash with 2 bed volumes of 1 N HCl
2. Wash with 5 bed volumes of Milli-Q water
3. Wash with 2 bed volumes of 1 N NaOH
4. Wash with Milli-Q water until the column washes reach neutral pH (many bed volumes!).

Using Chelex resin with dialysis to decalcify buffer solutions²

1. Dialysis tubing is boiled four times in Milli-Q water prior to adding neutral Chelex 100 resin to the bag.
2. The dialysis bag is then placed in the plastic container with the buffer solution.
3. Shaking or inverting several times a day for a week provides a calcium free solution.

Note:

Be prepared for the resin to expand and contract dramatically with changes in pH. This can affect the flow rate in your column, for example, if you pack the column before neutralizing the resin.

References

1. BioRad Product Literature

2. Thulin, E. (2001) Purification of Recombinant Calbindin D_9k. In: Methods in Molecular Biology, Vol. 172, pp. 175-184, Calcium-Binding Protein Protocols, Volume 1, Reviews and Case Studies (H. J. Vogel, ed.), Humana Press, Inc., Totowa, NJ