Gel filtration markers kit for protein

The molecular weights of the proteins of interest were determined in comparison to the protein standard, Gel Filtration Markers Kit for Protein (Molecular Weights 12,000–200,000 Da; Sigma-Aldrich). The relevant proteins and the protein standards were separated on a Superose^™ 6 Increase 10/300 GL column (GE Healthcare) connected to the ÄKTA Pure 25 system (GE Healthcare) using 1.5 CVs of SEC buffer. The void volume (v₀) of the column and the elution volume (v_E) of each protein were determined, and the ratio (v_E/v₀) calculated. A standard curve was derived by plotting the v_E/v₀ against the MW of the reference proteins and used to estimate the molecular weight of CdaA_103–285, Pde1_53–657 and Pde2 in solution.

All samples were reconstituted with hemin as described in the second paragraph of 4.2. All spectra were obtained on the same day on a Superdex™ Increase 200 10/300 GL in 20 mM Tris-HCl, 50 mM NaCl buffered at pH 7.4. The flow rate used was 0.4 mL/min. After column equilibration, a standard calibration curve (Figure S6) was prepared using the Gel Filtration Markers Kit for Protein from Sigma-Aldrich. After reconstitution and desalting, 200 μM of HupZ and HupZ samples (1 mL) were injected onto the SEC column.