| Method |
The concentration of each purified protein was determined by two independent methods. The amount of proteins applied to generate standard curves was calculated based on mean of the two values. Bradford-assay (Bio-Rad), DC protein-assay (Bio- Rad), and BCA-assay (Sigma) were performed according to the manufacturers’ manual with bovine serum albumin as a standard. The concentration of His6-Nat1 was determined with the DC proteinassay (Bio-Rad), which can be performed in the presence of detergent. The concentrations of His6- Asc1, His6-Ard1 and His6-Ssb1 were calculated from UV-absorption at 280 nm using molar extinction coefficients at 280 nm: e(His6-Asc1) = 71056 M-1 cm-1, e(His6- Ard1) = 23506 M-1 cm-1, and e(His6- Ssb1) = 19099 M-1 cm-1. Absorption at 280 nm could not be applied as a method for protein quantification when purification was performed under denaturing conditions, as urea interfered with measurements. |