Figure 5.

Sample small scale purification of a two subunit complex. The HIS-tagged A. thaliana Gcn5 deletion construct was coexpressed with an Ada2 deletion construct. Equivalent volumes of the whole cell extract (after sonication), extract pellet, extract supernatant and Talon flow through (what did not bind to the Talon resin) are shown in lanes 1 through 4 respectively. In this experiment, 5 ml of the extract supernatant was incubated with 0.5 ml of washed Talon metal affinity resin. Note that cell lysis of this extract was incomplete given that significant amounts of all proteins were found in the pellet fraction. Since most E. coli proteins are soluble, one normally expects relatively little protein in the pellet fraction. Also note that the tagged Gcn5 and untagged Ada2 proteins bound efficiently to the Talon resin as judged by the relative absence of these proteins in the Talon flow through fraction. Elution using three 0.5 ml fractions of solution containing 100 mM imidazole releases the bound Ada2/Gcn5 complex, predominantly in fraction 2 (lanes 6 through 8). The asterisk marks a known E. coli contaminant, EF-Tu, found in many of our preparations of HIS-tagged protein complexes purified over Talon resin (Barrios et al., 2007). Molecular weight markers are shown in lane 8, with their molecular weights in kilodaltons shown to the right of the gel.