Figure 2

Analysis of the purified PFV RNase H-(Q⁵⁹¹-N⁷⁵¹). (A) SDS-PAGE of the purified PFV RNase H-(Q⁵⁹¹-N⁷⁵¹). PFV RNase H-(Q⁵⁹¹-N⁷⁵¹) was purified via Ni-affinity chromatography, TEV cleavage followed by a second Ni-affinity column to remove the 6His-GB1 tag. The flow through was concentrated and analyzed by a 19% SDS-PAGE and Coomassie staining. Lane M, molecular weight standard, the apparent molecular weights are indicated on the left. The arrow indicates the band of PFV RNase H-(Q⁵⁹¹-N⁷⁵¹) of which different amounts were loaded. (B) Size exclusion chromatography of PFV RNase H-(Q⁵⁹¹-N⁷⁵¹) using an S75 HR 10/30 column. The run was performed with 50 nmol of purified PFV RNase H-(Q⁵⁹¹-N⁷⁵¹) in 50 mM Na-phosphate buffer, pH 6.8, 100 mM NaCl and 0.5 mM DTT. The inset shows the best fit to the data obtained for the molecular masses of the standard proteins (open circles), which was used for the determination of the molecular mass of PFV RNase H-(Q⁵⁹¹-N⁷⁵¹) (closed circle).