probond ni-nta resin column  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: CvfA Protein and Polynucleotide Phosphorylase Act in an Opposing Manner to Regulate Staphylococcus aureus Virulence *

doi: 10.1074/jbc.M114.554329

Figure Lengend Snippet: Purification of S. aureus wild-type PNPase and mutated PNPases. A, domains of PNPase are schematically presented. PNPase contains two catalytic domains named PH-1 and PH-2 (58) and two RNA binding domains (59, 60). B, S. aureus PNPase fused with His6 was overproduced in E. coli and purified by ammonium sulfate precipitation and Ni-NTA column chromatography. Fractions of ammonium sulfate precipitation (29 μg) and Ni-NTA column chromatography (5 μg) were analyzed by SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue. Poly(A) polymerization activity of each fraction is presented in Table 4. C, poly(A) polymerization activity of purified PNPase was measured at 37 °C for 15 min using ADP as a substrate. Vertical axis represents the amount of ADP incorporated into poly(A), and horizontal axis represents the amount of added PNPase protein. D, mutated PNPases were purified by the same method for wild-type PNPase. Purified proteins (1 μg) were analyzed by SDS-PAGE stained with Coomassie Brilliant Blue. E, poly(A) polymerization activities of mutated PNPases were measured using the same method as for wild-type PNPase. F, phosphorolytic activities of wild-type PNPase and mutated PNPases were measured at 37 °C using poly(A) as a substrate.

Article Snippet: The resulting precipitate was dissolved in buffer A (50 m m Tris-HCl (pH 8.0), 500 m m NaCl, 20% glycerol, 1 m m imidazole) and subjected to a Ni-NTA column (ProBond Resin, Invitrogen).

Techniques: Purification, RNA Binding Assay, Column Chromatography, SDS Page, Staining, Activity Assay