Ampholine

Bier, M Mosher, RA Palusinski, OA, Computer Simulation and Experimental Validation of Isoelectric Focusing in Ampholine-Free Systems, Journal of Chromatography 211, 313, 1981. Bier, M Palusinski, OA Mosher, RA Saville, DA, Electrophoresis Mathematical Modeling and Computer Simulation, Science 219, 1281, 1983. 

The immunoglobulin fraction from each bleed was purified by use of a recirculating isoelectric focusing (RIEF) technique (Bier et al. 1979). The whole serum was diluted 1 3 with urea, to yield a final urea concentration of 3 M, and then desalted by electrodialysis. The urea was added to prevent precipitation under hypotonic conditions. Ampholine (1 percent w/v, pH 3.5 to 10, LKB... 

A pH gradient is produced by incorporating a mixture of Ampholines with appropriate p/ values in either a polyacrylamide slab or column. An acid is used at the anode and an alkali at the cathode, the pH of these being approximately the same as the pi values of the two extremes of the Ampholine range. Phosphoric acid and sodium hydroxide are suitable for wide range separations while various amino or carboxylic electrolytes are used for intermediate pH ranges. 

The prepared Ampholine gel is set up in the tank and thick filter paper strips are soaked with either the anodic or cathodic electrolyte and placed along the appropriate edge of the gel. The samples may be applied either to small filter paper squares laid on the surface of the gel or, for bulk preparative work, incorporated in the gel. The appropriate voltage is applied through terminals attached to the electrode wicks and after about 30 min can be switched off to permit the removal of the sample filter papers before continuing the separation. 

Proteins or antibodies (36 pg) were mixed with ampholine pH 3.5—9.5 (final concentration of 5%, Amersham Biosciences, distributed by GE Healthcare, Uppsala, Sweden), p7 markers (Bio-Rad, Hercules, CA), and hydroxypropyl methyl cellulose (final concentration of 0.2% HPMC, Sigma-Aldrich, St. Louis, MO). The final protein concentration was 0.3mg/mL. Figure 17 shows a schematic of the sample preparation. The mixture was mixed thoroughly and was introduced to the capillary (eCAP neutral-coated, 50 micron X 30 cm, Beckman, Fullerton, CA) by hydrodynamic injection. Injections were performed using 20 psi for 99 s. The solution was then separated under an electric field of 25 kV for 10 min. The focused protein was then pushed/pulled out of the capillary through a mobilization process using the cathodic mobilizer (Bio-Rad, Hercules, CA). 

Tests of Purity, Isoelectric focusing (lEF) in a 0.5-mm thick horizontal slab gel was performed with LKB pH 7-9 ampholyte (Ampholine 1809-136) (5). Electrophoresis was run at 10°C for 6 h at a constant voltage of 1800 V. Protein was visualized using silver stain (9) or Sigma Coomassie Brilliant Blue G-250. 

Rat brain microsome preparations were conveniently stored at -10C with retention of enzyme activity. Solubilization with Triton X-100 appears to be effective and the solubilized enzyme preparation, after filtration once with Amicon XM-300 diaflo membrane, was introduced into an isoelectric focusing column (LKB) with an ampholine pH range of 3.5-10. 

Righetti, P.G., Brown, R. R and Stone, A. L. (1978). Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focussing. Biochim. Biophys. Acta 542, 232-244. 

CM cellulose Ampholine (pH 4-6) Human serum albumin, a-fetoprotein 35... 

Gel electrophoresis was performed in barbital buffer29 of pH 8.3, and gel isoelectrofocusing was performed in ampholine-sucrose solution of pH 5 to 8 gradient.28 Gels were removed from the apparatus at completion of... 

Figure 11.3. Typical Ampholine component.6 The pi values are varied by varying the number and type of the R4N+ and RCOO groups.

Isotachophoresis is similar to isoelectric focusing but includes the use of additional ampholines or multiphasic buffer systems to act as spacers to improve the separation and resolution. Each of these techniques can be run using columns, tubes, thin layers, or slabs and are comprehensively dealt with by Andrews (1981). 

CL Catsimpoolas, N., and Keeney, J., Analytical isotachophoresis of human serum proteins and ampholine spacers. Biochim. Biophys. Acta 285, 287-292 (1972). 

H13. Hjalmarsson, S.-G., Preparative isotachophoresis—the effect of using ampholine of different pH ranges as spacer ions in the fractionation of serum proteins. Sci. Tools 22, 35-38 (1975). 

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