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Isotopic labeling of recombinantly expressed proteins is generally required for investigation by modern nuclear magnetic resonance (NMR) methods. Purification strategies of the labeled proteins often include the use of a polyhistidine affinity tag (His-tag) and immobilized metal ion affinity chromatography (IMAC). Described herein are rapid and inexpensive qualitative and quantitative assays to determine the concentration of paramagnetic Ni 2+ in protein samples purified by IMAC. Both qualitative and quantitative colorimetric methods detect the amount of Ni 2+ via the color change produced when a [Ni(PAR) n ] 2+ (PAR=4-(2-pyridylazo)resorcinol, n=1, 2) complex is formed. The qualitative assay provides a rapid visual test for the presence of Ni 2+ in the low micromolar range in a sample of interest. The usefulness of the spectroscopic quantitative assay is illustrated by: (i) detecting a 12μM Ni 2+ contamination in an NMR sample containing 950μM of the 7.5kDa α 3 W protein purified by a standard His-tag Ni 2+ /IMAC approach and (ii) showing that the 15 N-HSQC spectrum of the α 3 W NMR sample, containing 1 paramagnetic Ni 2+ ion per 80 protein molecules, displays clear line broadening of both water and protein spectral lines. We also (iii) measured Ni 2+ release during the equilibration, wash, and elution steps of three commonly used Ni 2+ /IMAC resins when following manufacturer's protocols. The concentration of Ni 2+ detected in elutes of the three resins ranged from 2μM to nearly 1mM.

methods in enzymology on cd-rom/methods in enzymology

A Quick and Colorful Method to Measure Low-Level Contaminations of Paramagnetic Ni2+ in Protein Samples Purified by Immobilized Metal Ion Affinity Chromatography