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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Biochim Biophys Acta. 2010 Oct 26;1812(4):544–548. doi: 10.1016/j.bbadis.2010.10.010

Figure 3. Iron loading-induced precipitation of ferritin heteropolymers containing Mt-FTL subunits.

Figure 3

Ferrous ammonium sulphate was added to separate samples of heteropolymeric (Mt-FTL/FTH1, Wt-FTL/FTH1, and Mt-FTL/Wt-FTL) or homopolymeric (Mt-FTL and Wt-FTL) apoferritins, and incubated for 2 h. The protein concentration was 1 μM and iron concentrations were 0, 1, and 3 mM. Samples were centrifuged for 15 min at 10,000 g in order to separate soluble from aggregated protein. In contrast to the solubility of Wt-FTL/FTH1 heteropolymers, precipitation of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers was observed at 3 mM iron. (a) Soluble fractions of Mt-FTL/FTH1 and Wt-FTL/FTH1 heteropolymers were characterized by 3–8% native PAGE and stained with Coomasie blue. (b) The insoluble fraction was resuspended in Laemmli sample buffer, resolved by SDS 12% PAGE, and blotted with antibodies against FTH1 and Mt-FTL. (c) Mt-FTL/Wt-FTL heteropolymers and the corresponding homopolymers were iron loaded and electrophoresed as in (a) above, showing precipitation for hetero- or homopolymers containing Mt-FTL. (d) The insoluble fraction from Mt-FTL/Wt-FTL was resuspended, electrophoresed, and blotted as in (b) above, with antibodies against Wt-FTL and Mt-FTL.