Electrophoretic mobility shift assay of zinc finger proteins: Competition for Zn2+ bound to Sp1 in protocols including EDTA

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA–protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn2+ and readily competes with zinc finger peptides for Zn2+ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein–DNA adducts. This paper examines the chemistry that permits the detection of zinc finger–DNA complexes in the presence of EDTA, using Zn3-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1–GC1 was shown to depend on three properties: the inertness of Zn-Sp1–GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn3-Sp1 under the conditions of the assay that permit some Zn3-Sp1–GC1 to form. Inquiring about the mechanism of stabilization of Zn3-Sp1 by GC1, EDTA readily reacted with Zn3-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn3-Sp1 failed to compete for zinc bound in the Zn3-Sp1–GC-1 adduct. On the basis of these, other results indicated that the stability of Zn3-Sp1–GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.

Graphical AbstractEDTA inactivates Zn3-Sp1 DNA binding; yet EDTA is found in commercial kits including those used for electrophoretic mobility shift assays (EMSA) that measure the formation of protein–DNA adducts. We conclude that zinc finger protein binding in the EMSA rests on fortuitous chemical properties that may vary with the structures involved.Figure optionsDownload full-size imageDownload as PowerPoint slide