Methodology

The structure of carrier ampholytes (CA) and their general properties are illustrated in Figure 2. CAs are oligoprotic amino carboxylic acids, each containing at least four weak protolytic groups, at least one being a carboxyl group and at least one a basic nitrogen atom, but no peptide bonds. In a typical synthesis, a mixture of oligoamines (four to six nitrogens in length, linear and branched) reacts with an a-^-unsaturated acid (typically acrylic or itaconic acids), at a nitrogen-carboxyl ratio of 2 : 1.

The mechanism of developing a pH gradient in IEF is illustrated in Figure 3. Before passage of the current, the column is at constant pH (Figure 3A) and the multitude of amphoteric buffers is randomly distributed, resulting in a reciprocal neutralization. However, each individual CA species will have its own titration curve (see Figure 2, lower left side) defining different mobilities in the electric circuit. After starting the experiment the different CAs will migrate at different velocities in the column, the most acidic and most basic compounds being the fastest moving ions. As a result of this sorting process, a pH gradient will form, sigmoidal at first (Figure 3B), with an uneven voltage gradient. After 1 h, the various CA buffers will have separated further, and at this point an almost linear pH gradient has been established

Figure 2 Composition of Ampholine. On the upper left side a representative chemical formula is shown (aliphatic oligoamino oligocarboxylic acids). On the lower left side, portions of hypothetical titration curves of carrier ampholytes are depicted. Right: different pH cuts for wide and narrow range ampholytes (by permission of LKB Produkter AB).

Figure 2 Composition of Ampholine. On the upper left side a representative chemical formula is shown (aliphatic oligoamino oligocarboxylic acids). On the lower left side, portions of hypothetical titration curves of carrier ampholytes are depicted. Right: different pH cuts for wide and narrow range ampholytes (by permission of LKB Produkter AB).

which spans the pH range defined by the pis of the Gaussian profiles. Now the system has achieved ampholytes (Figure 3C). After 1.5 h the CAs have a steady-state, i.e. a balance between electrophoretic separated into symmetrical zones with overlapping transport and diffusion away from the pi, and no

Figure 3 Calculated time development of a focusing process involving 10 ampholytes in a closed vessel. The p/s of the ampholytes are evenly distributed in the pH 8.0-8.9 range. The initial distribution of the amphoteric buffers is indicated in (A). The calculation was performed assuming a constant voltage (100 V cm-1) across the system. The anode is positioned to the right in the diagrams. Each x-axis represents the distance from the cathode on the same scale as in (D). (Reproduced with permission from Schaefer-Nielsen, 1986.)

Figure 3 Calculated time development of a focusing process involving 10 ampholytes in a closed vessel. The p/s of the ampholytes are evenly distributed in the pH 8.0-8.9 range. The initial distribution of the amphoteric buffers is indicated in (A). The calculation was performed assuming a constant voltage (100 V cm-1) across the system. The anode is positioned to the right in the diagrams. Each x-axis represents the distance from the cathode on the same scale as in (D). (Reproduced with permission from Schaefer-Nielsen, 1986.)

further mass transport is expected (Figure 3D). As long as the local concentration of the different CA species does not change the slope of the pH gradient will be kept constant with time. Proteins will keep migrating against this CA distribution profile eventually reaching their pi position.

By and large, most analytical IEF runs are performed in horizontal chambers: the polyacrylamide gel slab rests on a cooling block [generally made of glass or coated aluminium or even beryllium oxide (used as the heat shield of the space shuttle)]. This horizontal configuration allows one to dispose of electrode reservoirs and of all the hydraulic problems connected with vertical chambers (tight seals, etc.): in fact, anolyte and catholyte are soaked in filter paper strips resting directly on the open gel surface. In addition, most modern chambers contain a cover lid with movable electrodes which can be adjusted to any gel length (generally from 10 to 25 cm electrode distance). Since thick gels (e.g. 2 mm thick) generate thermal gradients through the gel thickness, resulting in skewed zones (essentially all horizontal chambers have cooling only on one gel face) ultrathin gels (0.2-0.5 mm) supported on a reactive polyester foil (Gel Bond PAG) are preferred today.

Solar Panel Basics

Solar Panel Basics

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