After analysis of the highest calibration sample (0.9 μM), a blank was injected (mobile phase A or TBS buffer) and the S1P peak measured.
In addition, 2D NOESY was collected for an unlabeled ZFD prepared in 100% D2O buffer, and the NOE cross-peaks were manually assigned mainly involving aromatic side chains.
The ammonium sulphate pellet was suspended in appropriate volume of Buffer A and loaded onto 8 ml Mono-Q column (Amersham Life Sciences) equilibrated with 10 column volumes (80 ml) of Buffer A. Following a wash with 3 column volumes of buffer A, the protein of interest was eluted with 10 column volumes of Buffer B (Buffer A+1M NaCl-Linear gradient) and the peak fractions were pooled.
The protein was eluted by running one column volume of R plus 1 M KCl buffer through the column and the peak fractions were identified by SDS-PAGE.
Fractions containing Saw1 (500 1,000 mM imidazole) were applied onto a 0.5 ml MonoS column (GE Healthcare), and eluted using 200 1,000 mM KCI in buffer T. The peak Saw1 fractions (550 1,000 mM KCI) were concentrated to 3 µg/µl in a Vivaspin-2 concentrator.
To further improve the purity of the protein, the concentrated protein was loaded onto a Superdex-200 gel-filtration column equilibrated with Buffer C. The peak fractions from the elution were pooled and tested by SDS-PAGE or Western blotting.
The results indicated that, in pH 7.1 Tris HCl buffer solution, the peak current was linear with the concentration of thrombin in the range of 0.8 15 nM with a detection limit of 0.2 nM.
After the column was washed with five column volumes of buffer B, the enzyme was eluted with a linear gradient of 0 0.5 M imidazole in buffer B. The peak of enzyme activity eluted at an imidazole concentration range of 0.06 0.27 M.
As the electrochemical probe, quinone derivative can generate a pair of redox waves in aqueous buffer and its peak current is correlated with the activity of NQO1, so an electrochemical method to determine the enzyme activity can be proposed.
(b) The air spring shock absorber is an effective buffer to reduce the peak load and the peak torque, and have a longer serve lives.
