2. Stacking gel: For casting of the stacking gel, a monomer concentration of 2.5% T with a crosslinker concentration of 20% C was used. Fifty ml of leading electrolyte were mixed with 1.0 g acrylamide and 0.25 g Bis. After degassing, 30 l of TEMED and 300 l of 10% ammonium persulfate solution were added. The stacking gel solution was cast on the separating gel to a height of 1 cm. The stacking gel was overlayed with water-saturated 2-butanol. After polymerization the butanol was removed and the gel surface was rinsed with water. The gel was used for running after about 15 h in order to obtain complete polymerization.
The leading electrolyte (gel and lower anode buffer chamber) was 0.05 M HCl, pH 8.0 (the pH was adjusted by Tris); the terminating electrolyte (upper cathode buffer chamber) was 0.153 M 6-aminocaproic acid, pH 8.9 (Tris); the elution buffer was 0.02 M ammonium acetate, pH 8.0 (Tris).
The samples analyzed were selected fractions from gel chromatography that had been concentrated by freeze-drying. Carrier ampholytes were added to a sample solution of less than 3.0 mg (0.500 ml sample volume, pH 8.0, + 0.1 ml carrier ampholyte, pH 5 7, + 0.1 ml carrier ampholyte, pH 6 8, + 0.1 ml carrier ampholyte, pH 7 9). Bromophenol blue was used as a marker. The sample was carefully loaded on the surface of the stacking gel.
The operating conditions were:
Initial voltage 400 V constant
Initial current 6 - 12 mA
Final voltage 400 - 250 V
Final current 12 mA constant