DGAT1 protein was enriched by Nickel-affinity chromatography (GE Health) and eluted with a step gradient of imidazole at 4 °C.
A step gradient of 20, 40, 60 and 80 % MeOH in water (v/v), each 700 mL, has been used at a flow rate of 20 mL/min.
Elution of the alkaloids was effected using a step gradient of CH2Cl2-isopropanol (97 3), CH2Cl2-isopropanol (93:7), and then CH2Cl2-isopropanol (75:25).
The target protein binds at specific salt conditions and can be eluted by a step gradient decreasing the pH value and reducing the ionic strength.
The mobile phase was a step gradient of a sodium citrate solution, pH 5.28, at a flow rate of 0.4 ml/min, and the column was maintained at 50°C, also according to manufacturer's procedure.
During subculture, different concentrations of NaCl solution were applied to the BG11 medium in a step gradient (0, 18, 24, 48, 60, 90, and 96 g l−1 NaCl).
Trypsin-like enzymes were then eluted from the column using a step gradient of NaCl with different concentrations ranging from 100 to 500 mM NaCl in the starting buffer.
The enzyme was purified from the supernatant by immobilized metal ion chromatography using Ni-loaded IMAC Sepharose 6 Fast Flow GE Healthcaree) and a step gradient of imidazole as eluent.
The supernatant was layered on top of a step gradient consisting of 1 mL 45%, 33%, and 15% (w/w) sucrose, respectively, and then centrifuged for 2 h at 70,000 × g.
BuOH Soluble extract (56 g) was applied to a Diaion HP20 column (10 × 200 cm) with a step gradient elution of EtOH-H2O (v/v 0 1, 4 6, 9.5 0.5) to provide three factions: A1, A2 and A3.
An SFE trap system using C1 stationary phase connected to a C18 analytical HPLC column enabled the SFE LC MS MS analysis of three polyether ionophore antibiotics in water using a step gradient.
