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The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension.
The IPG strips were then placed onto a 12.5% SDS-PAGE gel and the second dimensional separation was performed in two steps at 10°C: 80 V/gel for 30 min and 120 V/gel until the tracking dye reached the bottom of the gels.
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Each fraction from the first dimension (column 1) was stored in the parallel sampling loops and further introduced in the second dimensional column alternatively every 2.5 min. In total, 12 fractions were collected, and thus, 30 min of the first dimension was conducted for comprehensive two-dimensional analysis.
In Fig. 7, the horizontal axis x represents the first dimensional value and the vertical axis y represents the second dimensional value in the 2-dimensional feature matrix.
The second dimensional SDS-PAGE was performed in 12% acrylamide gels.
The selected spots were cut from the second dimensional gel stained with SYPRO-Ruby.
The second dimensional electrophoresis was carried out in a BioRad Criterion electrophoresis cell system.
The second dimensional separation was carried out on the custom-made 12% SDS-polyacrylamide gels and a Hoefer DALT electrophoresis system (Amersham Biosciences).
The second dimensional electrophoresis was performed at 80 V for 4 h, 100 V for 4 h, and 120 V for 2 h.
The second dimensional separation was performed using an Ettan™ DALT system (Amersham) at 0.5 W/gel and 16°C overnight, followed by 17 W/gel for 3 h.
He left a body of work comprising more than a hundred technical papers along the lines of "Reliable Circuits Using Less Reliable Relays," as well as others, not quite so influential, like "Scientific Aspects of Juggling" and "The Fourth-Dimensional Twist, or a Modest Proposal in Aid of the American Driver in England".
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