Additional file 6: Figure S5. of Structural basis for potency differences between GDF8 and GDF11

Purification and quantification of GDF8/GDF11 chimeric ligands. A, B Representation of purified protein from selected GDF8/GDF11 chimeras under non-reduced (A) and reduced (B) conditions (4–15% gradient gel). Chimeras or empty vector control were produced transiently using HEK293T cells and purified using size exclusion chromatography. The resultant peak containing the prodomain:mature ligand complex was pooled and concentrated. For empty vector control, corresponding fractions from a similar retention volume were pooled. The lane labeled “pro domain + mature” serves as a control for the molecular weight of purified wt GDF8 prodomain and wt GDF8 mature ligand. Note the expected changes in mass of the mature ligand (blue arrows) under non-reducing (dimer) and reducing (monomer) conditions while the prodomain mass is relatively unaffected (gray arrow). Protein is visualized by colloidal Coomassie stain. To ensure that comparable amounts of each GDF8/GDF11 chimeric protein were being administered in the cell-based assays, we first normalized protein concentrations based on the amount of dimer present in a non-reduced SDS-PAGE gel stained with colloidal Coomassie. The samples were then normalized and reexamined by SDS-PAGE gel under non-reducing and reducing gel. The subsequent bands were quantified (bottom, below gel) under non-reduced (A) and reduced (B) gels using ImageJ showing that the protein concentrations were indeed normalized. 500 ng of recombinant GDF8 prodomain and purified GDF8 mature were loaded for reference. (TIF 3694 kb)