Experimental Issues Regarding Using Metal Chelators

In most PLIMSTEX experiments, the total ligand concentration is used in the curve fitting. The determination of free ligand concentration is not required because the relationship between the free and total ligand concentrations is resolved in the modeling procedure [24]. For the ras titration, however, we had no Mg2+-free ras-GDP stock but only a limited amount of ras-GDP-Mg2+ stock solution (1.5 mM ras, 10 mM MgCl2, in 64 mM Tris-HCl, 10 mM MgCl2, 1 mM sodium azide, 1 mM DTT buffer, pH 7.6). After diluting by 100 times with 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and 0.1 M KCl, pH 7.4 buffer, there was still excess of Mg2+. To conduct the Mg2+ titration of free ras-GDP, we added EDTA to control free [Mg2+] in solution {free [Mg2+] was calculated using a WebChelators program (Webmaxc Standard) on the internet [43, 44]}.

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