Soluble fraction was subjected to immobilized metal ion affinity chromatography (IMAC), and the recombinant protein was eluted by buffer A with a gradient of 10 to 300 mM imidazole.
Concentrated protein was successively applied to Resource Q and Resource S 1 mL columns (GE Healthcare), and the protein was eluted from the column using buffer E with a gradient of NaCl from 0 M to 2 M. Finally, the protein was loaded onto a Superdex 75 10/300 GL column (GE Healthcare) in buffer F (10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5).
Bound proteins were eluted in the same buffer, with a continuous NaCl gradient from 0 to 0.45 M at a flow-rate of 1 ml.min-1.
With the full buffer model, a gradient scheduling algorithm exhibits a trade-off between average user throughput and the user throughput at 5% outage.
The results also show that with the full buffer model a gradient scheduler exhibits a trade-off between average user throughput and the user throughput at 5% outage, but it does not when the more realistic finite buffer is used.
After desalting with a Sephadex G25 column, the enzyme was applied to a Source 15S column (GE Healthcare) in the same buffer, followed by elution with a gradient up to 1 M NaCl.
A gradient buffer with a decreasing concentration of denaturing agent was passed over the immobilized PrP for refolding.
In this system, DNA fragments are carried by a gradient buffer (with a 2% increase in buffer per minute) through a DNASep® Cartridge under isothermal (58°C and 55.6°C, respectively) conditions, with subsequent detection by absorbance at 260 nm.
The resulting supernatant was loaded onto two in line connected 1 ml HisTrap columns (GE Healthcare, Germany), washed with 80 ml buffer A and then eluted with a gradient from 0 to 100% over 80 ml of buffer B (0.3 M NaCl, 1 mM MgCl2, 0.5 M imidazole, 50 mM NaH2PO4, pH 7.5).
Cleared lysates were incubated overnight at RT with shaking, purified on Ni-NTA column, washed with a gradient of buffer G and buffer B (10 mM Tris pH 8.0, 100 mM Na2HPO4, 0.1 mM oxidized glutathione, 10 mM imidazole) at ratios of 6∶0, 5∶1, 4∶2, 3∶3, 4∶2, 5∶1 and 0∶6, respectively.
