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Comparative Study
. 2000:326:245-54.
doi: 10.1016/s0076-6879(00)26058-8.

Purification of proteins using polyhistidine affinity tags

J A Bornhorst  1 , J J Falke
Affiliations
Comparative Study

Purification of proteins using polyhistidine affinity tags

J A Bornhorst et al. Methods Enzymol. 2000.
No abstract available

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Figures

Fig. 1
Fig. 1
Models of the interactions between the polyhistidine affinity tag and two immobilized metal affinity chromatography matrices, (a) The nickel–nitrilotriacetic acid matrix (Ni+2–NTA) [from J. Crowe, H. Döbeli, R. Gentz, E. Hochuli, D. Stüber, and K. Henco, Methods Mol. Biol. 31, 371 (1994)]. (b) The cobalt–carboxylmethylaspartate matrix (Co2+–CMA) (from G. Tchaga, Clontech, personal communication). In both cases, the metal ion exhibits octahedral coordination by four matrix ligands and two histidine side chains, the latter provided by the polyhistidine affinity tag.
Fig. 2
Fig. 2
SDS–PAGE analysis of a representative polyhistidine-tagged protein purification using a nickel-nitrilotriacetic acid (Ni2+–NTA) matrix. The 42-kDa MAP-kinase protein ERK2 was affinity tagged at its N terminus with six histidines [D. J. Robbins, E. Zhen, H. Owaki, C. A. Vanderbuilt, D. Ebert, T. D. Geppert, and M. H. Cobb, J. Biol. Chem. 268, 5097 (1993)], and was isolated using the procedure detailed in the text. Shown are equal volumes of the cell lysate, the breakthrough material that failed to bind to the resin during the batch step, the wash material obtained after loading the resin into the column, and the eluate from the column. The 15% Laemmli gel was visualized by Coomassie staining.
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References

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