ANALYTICAL BIOCHEMISTRY 236, 101–106 (1996) ARTICLE NO. 0137 Single-Step Synthesis and Characterization of Biotinylated Nitrilotriacetic Acid, a Unique Reagent for the Detection of Histidine-Tagged Proteins Immobilized on Nitrocellulose 1 Scott A. McMahan and Richard R. Burgess 2 McArdle Laboratory for Cancer Research, University of Wisconsin – Madison, Madison, Wisconsin 53706 Received October 19, 1995 derivative has become prominent. Its main application Using a one-step reaction, a bifunctional compound has been the rapid purification of a protein cloned into was synthesized for detecting histidine-tagged pro- a vector providing high levels of expression. This vector teins immobilized on nitrocellulose. This compound is constructed to put 6 – 10 histidines at either terminus has a biotin as one functional group and a nitrilotria- of the protein. This histidine-tagged protein is bound cetic acid as the other. The nitrilotriacetic acid is used by the immobilized metal, while other proteins are to chelate a Ni(II) ion at four of its six coordination washed off the column. The purified protein can then sites. The remaining two sites are available for binding be eluted (2, 3). to a histidine tag. The biotin functional group can then In addition to providing a means of purifying protein be detected using a streptavidin – horseradish peroxi- from a whole cell lysate, our lab has shown that tagging dase conjugate and chemiluminescence. Using this bi- a protein with a run of histidines can be useful for otinylated nitrilotriacetic acid, it is possible to detect epitope mapping of monoclonal antibodies (Rao, L., less than 0.11 pmol of histidine-tagged Escherichia coli Jones, D. P., Nguyen, L. H., McMahan, S. A., and Bur- RNA polymerase s 70 subunit. This reagent is also able gess, R. R., in preparation). This procedure works best to specifically detect His-tagged s 70 from a whole cell when an antibody is available that has previously been lysate following SDS – PAGE and transfer to nitrocellu- mapped very close to the tagged terminus. lose. The reagent can be dissociated from the His- A more general, less time-consuming, and less expen- tagged protein at pH 4.8 and the blot can be reprobed sive solution would be to detect the His-tag region it- with a monoclonal antibody for detection of different self. Because the histidine tag is relatively nonimmu- proteins on the same blot.  1996 Academic Press, Inc. nogenic, we took a chemical and not an immunological approach. We reasoned that a compound that had the Since its introduction by Porath et al. in 1975 (1), same functional group as the nickel chelate column metal affinity chromatography has become increas- would still be able to bind to the histidine-tagged pro- ingly popular. A wide variety of metals and proteins tein immobilized on nitrocellulose. In addition, if this have been used in these systems, but recently the use of compound were biotinylated, we could then use a strep- nickel(II) ions chelated by a nitrilotriacetic acid (NTA) 3 tavidin-conjugated enzyme for detection (4). The syn- thesis we used to make such a compound is efficient, 1 This work was supported by NIH Grants GM28575 and CA07175. inexpensive, and operationally simple. The sensitivity 2 To whom correspondence should be addressed. Fax: (608) of this compound is comparable to many monoclonal 262-2824. antibodies. The compound is selective enough to pro- 3 Abbreviations used: BNTA, biotinylated nitrilotriacetic acid; NTA, nitrilotriacetic acid; SDS, sodium dodecyl sulfate; SDS- vide easy identification of the recombinant His-tagged PAGE, SDS polyacrylamide gel electrophoresis, Mops, 3-(N-mor- protein on a Western blot even if the starting material pholino)propanesulfonic acid; PBS, phosphate-buffered saline, is as complex a mixture as whole cell lysates. 137 mM NaCl, 2.68 mM KCl, 8.06 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 ; TBST, Tris-buffered saline–Tween 20, 10 mM tris(hydroxy- methyl)-aminomethane hydrochloride, pH 7.6, 150 mM NaCl, 0.1% Tween 20; MBST, Mops-buffered saline–Tween 20, 20 mM Mops, min; ECL, enhanced chemiluminescence; EDTA, ethylenedi- aminetetraacetic acid. pH 7.9, 150 mM NaCl, 0.1% Tween 20; BSA, bovine serum albu- 101 0003-2697/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.