title: Artifact-inducing enrichment of ethylenediaminetetraacetic acid and ethyleneglycoltetraacetic acid on anion exchange resins.
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 sample 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 (0.1-0.5mM) 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 concentration in downstream assays.
authors.affiliation: Cellular and Molecular Biology Program, University of Wisconsin - Madison, Madison, WI 53706, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Chumanov
authors.forename: Richard R
authors.initials: RR
authors.lastname: Burgess
mesh_terms: D000837:Anion Exchange Resins / Q000737:chemistry*; D016477:Artifacts; D002614:Chelating Agents / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004492:Edetic Acid / Q000737:chemistry*; D004533:Egtazic Acid / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding
chemical_list: D000837:Anion Exchange Resins; D002614:Chelating Agents; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D004492:Edetic Acid