Acquity uplc protein beh sec column

Plasma (45 μL) was incubated with probe B-1 (20 μM; 3.2 μL of probe B-1 from a 300 μM stock solution in DMSO) or 3.2 μL of DMSO for 24 hours at 37°C. Before injecting onto the size exclusion column (Acquity UPLC protein BEH SEC Column, 200Å, 1.7 μm, 4.6 mm × 150 mm, Waters), each plasma sample was filtered through a P30 gel filtration column (Bio-Spin Columns with Bio-Gel P-30) to remove excess unbound peptide and preserve the lifespan of the SEC column (final volume = 50 μL). 5 μL of each sample was then injected onto the column and separated using a constant flow of 0.2 mL/min of 10 mM phosphate buffer, 1 mM EDTA, 100 mM KCl, pH 7.8, for 30 minutes. To account for plasma auto-fluorescence, the resulting signal from the samples with no probe B-1 (DMSO only) was subtracted from the signal from the corresponding sample with the probe B-1. At the start and the end of each sample set, four injections of different concentrations of fluorescein in the running buffer (freshly prepared each day) were analyzed by the same instrument, in order to make a standard curve for day-to-day comparison. All data were then normalized to the total protein concentration as calculated from the integrated area in the 280 nm absorbance channel. The results of the three groups were compared using a one-way analysis of variance (ANOVA) test.