In gel electrophoresis proteins are normally separated by charge, size, or shape. The aim of isoelectric focusing (IEF), for example, is to separate proteins according to their isoelectric point (pI), thus, according to their charge at different pH values. Here, a similar mechanism is accomplished in a commercially available electrophoresis chamber (cf. fig. Equipment) for separating charged biomolecules, for example, superoxide dismutase (SOD) or allergens, at constant pH conditions and different velocities of migration depending on different isoelectric points of zwitterions. The separated (metal) proteins elute sequentially, starting with the lowest (pI > 2–4) and ending with the highest pI (pI < 10.0) of the dissolved protein molecules to be analyzed.
Due to the specific properties of the prepared gel and electrophoresis buffer solution which is basic and contains Tris-HCl and NaN, most proteins of a biological system (e.g., Helicobacter pylori) are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field. In general, reaction equation (1) shows that the carboxyl side group of a proteinogenic amino acid is negatively charged, equation (2) that the amino side groups are electrically neutral under these conditions:
(1) R-COOH + OH → R-COO + HO
(2) R-NH + OH   →  R-NH + HO
At the anode,  electrochemically-generated hydrogen ions react with Tris molecules to form monovalent Tris ions (3). The positively charged Tris ions migrate through the gel to the cathode where they neutralise hydroxide ions to form Tris molecules and water (4):
(3) (HOCH)CNH + H  → [(HOCH)CNH]
(4) [(HOCH)CNH] + OH   → (HOCH)CNH + HO
Thus, the Tris-based buffering mechanism causes a constant pH in the continuous buffer system with a high buffer capacity.
At 25 °C Tris buffer has an effective pH range between 7.5 and 9.0. Under the conditions given here (addressing the concentration of buffer components, buffering mechanism, pH and temperature) the effective pH is shifted in the range of about 10.0 to 10.5. Native buffer systems all have low conductivity and range in pH from 3.8 to 10.2. Continuous native buffer systems are thus used to separate proteins according their pI.
Although the pH value (10.00) of the electrophoresis buffer does not correspond to a physiological pH value within a cell or tissue type, the separated ring-shaped protein bands are eluted continuously into a physiological buffer solution (pH 8.00) and isolated in different fractions (cf. fig. Electropherogram). Provided that irreversible denaturation cannot be demonstrated by an independent procedure, most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C. As the Joule heat and temperature generated during electrophoresis may exceed 50 °C, and thus have a negative impact on the stability and migration behavior of proteins in the gel, the separation system, consisting of the electrophoresis chamber, fraction collector and other devices, is cooled in a refrigerator at 4 °C, thus greatly reducing the risk of heat convection currents. Overheating of the gel is impeded by internal cooling circuit of the gel column as an integrated part of the electrophoresis chamber and by generating a constant power by the power supply (cf. fig. Equipment).