Artifact-inducing enrichment of ethylenediaminetetraacetic acid and ethyleneglycoltetraacetic acid on anion exchange resins Robert S. Chumanov a,b , Richard R. Burgess b,⇑ a Cellular and Molecular Biology Program, University of Wisconsin – Madison, Madison, WI 53706, USA b McArdle Laboratory for Cancer Research, University of Wisconsin – Madison, Madison, WI 53706, USA article info Article history: Received 22 October 2010 Received in revised form 20 January 2011 Accepted 20 January 2011 Available online 24 January 2011 Keywords: Anion exchange chromatography EDTA enrichment Purification artifacts EGTA abstract Multivalent metal chelators, ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA), are used extensively during protein purification. Both strong (Q) and weak (DEAE) anion exchange resins were found to adsorb surprisingly large quantities of EDTA and EGTA that elute from the resin at NaCl concentrations of approximately 240 mM (EDTA) and 140 mM (EGTA). The EDTA/EGTA elution and saturation parameters were determined for five commonly used anion exchange resins. The resulting concentration of eluted EDTA was 10- to 200-fold higher than that originally present in the sam- ple or in the mobile phase. Samples from fractions containing such a high concentration of EDTA were found to inhibit Mg 2+ -dependent polymerase chain reaction (PCR). EDTA binding to the anion exchange resins could saturate the resin, decrease its binding capacity, and displace weakly bound proteins such as green fluorescent protein (GFP). Several steps are suggested to minimize on-column EDTA concentration, including column equilibration in the absence of any EDTA, lower concentrations (0.1–0.5 mM) of EDTA, monitoring eluate absorbance at 280 nm as well as at 215 nm, adding EDTA back into fractions eluting before the EDTA peak, and performing blank column runs to control for the effect of changes in EDTA con- centration in downstream assays. Ó 2011 Elsevier Inc. All rights reserved. Ion exchange chromatography (IEC) 1 is a widely used separation technique for protein purification found not only in the research laboratory but also in large industrial settings. IEC is useful for frac- tionation of crude lysates during the early stages of protein purifica- tion as well as for removing trace contaminants during final polishing steps. The basis for chromatographic separation is the interaction of stationary phase with oppositely charged groups found on the protein. Two kinds of IEC stationary phases can be used: the negatively charged resin (cation exchange chromatogra- phy) or the more frequently used positively charged resin (anion ex- change chromatography [AEC]). The mobile phase contains salt ions that will compete with the protein for the binding to the stationary phase, depending on their relative affinities. The elution is per- formed by increasing the ionic strength (salt concentration) of the mobile phase; the increasing concentration of salt ions eventually causes the displacement of bound protein from the stationary phase. The amphoteric nature of proteins allows optimization of buffer parameters (e.g., pH, starting salt concentration) to increase efficiency of chromatographic separation [1]. The net ionic charge of the protein at a given pH dictates what stationary phase will bind the protein. In general, AEC is used at pH values above the tar- get protein’s pI, and cation exchange chromatography is used at pH values lower than the pI [1]. Most protein purification is performed at close to physiological pH to preserve the native conformation and enzymatic activity of the protein of interest. The IEC mobile phase is buffered appropriately for the intended pH of purification, and additives such as reducing agents, protease inhibitors, glycerol, and ethylenediaminetetraacetic acid (EDTA) are frequently added to preserve protein activity. EDTA is included in protein purification buffers because it che- lates both divalent and trivalent cations and prevents a number of deleterious effects during protein purification and storage. It de- creases the metal ion-dependent protease activity in crude protein extracts. It serves to decrease the metal ion-catalyzed oxidative damage to proteins; its presence stabilizes reducing agents such as dithiothreitol (DTT), allowing maintenance of reducing environ- ment during protein purification. EDTA chelates can also decrease the formation of disulfide bonds [2]. In some cases, both EDTA and ethyleneglycoltetraacetic acid (EGTA) are used as eluants during protein purification. EDTA can be used to elute proteins from resin 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.01.027 ⇑ Corresponding author. Fax: +1 608 262 2824. E-mail address: burgess@oncology.wisc.edu (R.R. Burgess). 1 Abbreviations used: IEC, ion exchange chromatography; AEC, anion exchange chromatography; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; EGTA, ethyleneglycoltetraacetic acid; TAP, tandem affinity purification; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; UV/Visible, ultravi- olet/visible; dH 2 O, deionized H 2 O; CV, column volumes; EGFP, enhanced green fluorescent protein; BSA, bovine serum albumin. Analytical Biochemistry 412 (2011) 34–39 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio