Labeled aptamers were separated from the unlabeled fraction and purified using a linear gradient from 5 to 30% ACN over 20 min.
This chromatography was performed using a linear gradient of 0.1% acetic acid acetonitrile from 10%to100%0% over 20 min at 60 ml min−1.
The analyses were done at a constant temperature of 37 °C and using a linear gradient system composed of A: 0.1% (v/v) formic acid in water, and B: 0.1% (v/v) formic acid in acetonitrile.
Cleaved MccB was eluted using a linear gradient of 0 1 M NaCl and was dialyzed into 50 mM Tris pH 8.0 and 1 mM DTT for co-crystallization screens.
However, the peak capacity obtained using a linear gradient is not much better than what can be obtained using conventional CE.
Components were separated using a linear gradient of acetonitrile (ACN) and FA in ultrapure water (flow rate 0.3 ml/min) according to the following scheme: 0 min=3% ACN, 0.1% FA; 3.5 min=35% ACN, 0.1% FA; 4.5 min=90% ACN, 0.1% FA; 5.5 min=3% ACN, 0.1% FA.
Next, peptides were separated on a C18 reversed phase analytical column (Acclaim PepMap RSLC, 75 µm × 15 cm, nanoViper, 2 µm 100 Å C18 particles; Thermo Scientific, USA) at 40 °C using a linear gradient of 5 35% acetonitrile 0.1% formic acid in 60 min at 600 nl min−1.
Peptide mixtures were trapped on a ReproSil C18 reversed phase column (1.5 cm × 100 μm) at a rate of 8 μL/min, separated using a linear gradient of 0 80% acetonitrile (in 0.1% formic acid) during 60 min at a rate of 200 nL/min using a splitter.
Combined fractions containing either malacidin A or B were subsequently cleaned up individually using preparative high-performance liquid chromatography (HPLC; XBridge Prep C18, 10 × 150 mM, 5 μM, Agilent HPLC System) using a linear gradient of 0.1% trifloroacetic acid acetonitrile from 30%to50%0% over 30 min at a flow rate of 4 ml min−1.
14-3-3 14-3-3 14-3-3 14-3-3r proteins by hydrophobic interaction chromatography on Phenyl Sepharose-6 Fast Floweregh Sub (GE Healthcare) using a linear gradient ofurthero 0 M NaCl in 50 mM sodium purifiede pH 7.0, 1 mM TCEP, followed by SEC.
The column was washed with the same buffer, and adsorbed enzyme was eluted using a linear gradient from 0 to 0.1 M phosphate buffer (pH 8.0).
