However, western blotting indicated that some target protein was lost during the wash and flow-through steps of affinity purification.
The first step (cation exchange) captures rhTyrRS while contaminants flow through, the second step (gel filtration) exchanges the buffer to PB, and the last step (anion exchange) removes the remaining process-related impurities.
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The flow through, first wash step, second wash step, last wash step, and elution fractions were analyzed by SDS PAGE.
After that, 200 μL 50 mM NH4HCO3 solution (pH = 7.8) was added and centrifuged at 14,000g for 15 min, discarded the flow-through repeated this step for twice.
The flow-through from this step was concentrated and loaded onto a Superdex 200 10/300 (GE Healthcare, Pittsburgh, PA) gel filtration column in the buffer 20 mM HEPES pH 8.0, 150 mM KCl, 5 mM DTT, 1 mM EDTA, 0.2% DM.
Both of the laminar and turbulent flows through facing step and corrugated channels are introduced.
The flow through from this step was collected for use in the small RNA extraction.
The uninfected Daphnia dynamics in our batch-type setup resemble those obtained in two-step flow-through chemostats [ 14, 48].
Although the flow-through from IMAC one-step phosphopeptide enrichment was discarded in most experiments, it was shown to contain a number of phosphopeptides that had a weak affinity to the chromatography matrix.
