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Separation of a cell lysate with a 60 min gradient showed extremely high peak capacities of 750 and above for a peptide and relatively homogeneous proteins.
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The injection volume was 5 mL, and the HPLC flow rate was 0.25 mL/min using the gradient shown in Table 1.
Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development.
They also show that while the Min gradient is independent of the chemical nature of the lipid bilayer, geometric cues play a rather critical role in its patterning.
Figure 5 shows the base-peak chromatogram for a 60 min gradient elution.
A 25 min gradient elution (from 5% to 95% mobile phase B) was used at 2.4 mL/min.
Data collection occurred over the entire 620 min gradient.
The first 10 fractions from SCX were analyzed using an 88 min gradient and the next 11 fractions were analyzed using a 55 min gradient, since the first 10 fractions had greater sample complexity, allowing for more peptide identifications (data not shown).
The mobile phase was A = water and B = acetonitrile, in a gradient flow: 1 min at 60%A/40%% B; to 2.5 min, gradient to 40%A/60%% B; to 4 min, gradient to 25%A/75%% B; to 5.5 min, gradient to 0%A/100%% B; and to 6.5 min, gradient to 60%A/40%% B with a 0.4 ml/min flow rate.
The Min protein oscillations and Min gradient formation were supported by both profiles.
The mobile phase was A = 50 mM CH3COONa, pH 6.3 with 0.04% TEA and B = acetonitrile with 0.04% TEA, in a gradient flow: 1 min at 96%A/4%% B; 5 11 min gradient to 89% A/11% B; 12 13 min gradient to 0%A/100%% B; and 14 min gradient to 96%A/4%% B with a 0.05-ml/min flow rate.
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