The column was equilibrated in running buffer (2 × PBS (phosphate-buffered saline)) and calibrated with different standard proteins: Tyroglobulin (660 kDa), Ferritin (440 kDa), Catalase (240 kDa), and BSA (bovine serum albumin) (67 kDa).
In the enhanced format the assay had limits of detection (LOD) of 3.7 pg ml−1 with standard in running buffer, and 15.4 pg ml−1 in a stripped human saliva matrix.
Twenty-four hours after transfection, the cell lysate was fractionated by 8% Native Gel in running buffer containing 1% sodium deoxycholate at 4°C, as previously described (Frieman et al., 2009).
Gels were pre-run without loading samples to gel in running buffer (pH 7.0) containing 2 mM sodium ascorbate for an hour to equilibrate gel before the electrophoretic separation.
In our trials, the column effluent was monitored at a flow rate of 0.35 mL/min in running buffer (20 mmol/L Tris-HCl, 50 mmol/L NaCl, pH 7.9).
All samples were in running buffer to minimize bulk effects.
The eluent was then evaporated to dryness at 60°C, and the dried extract was redissolved in running buffer.
Helicase samples (10 µM) were prepared in running buffer and were injected at 20 µl/min across the sensor surface.
Purified Ubc13 was maintained at 400 nM in running buffer (10 mM HEPES, 0.15 M NaCl, pH 7.4).
Chemokines were suspended at 0.1 µg/ml in running buffer and for binding experiments and all protein solutions were filtered through a 0.22 µm filter.
Protein was diluted in running buffer containing HBS buffer (Biacore), 0.005% (w/v) p-20 (Biacore), 2 mM MgCl2, and 2 mM ATP (pH 8.0).
