Influence of Hydrogen Ion Concentrations on the Protein Mobility in Native-PAGE Buffers

The study demonstrates the effect of hydrogen ion concentrations (pH) of Tank Buffer (TB) and resolving gel buffer (RGB) in a native-PAGE system on protein samples. A pH range of 7.8 to 9.3 for RGB and 8.3 to 9.3 for TB were used. Proteins in the samples under native-PAGE at varying pH of RGB such as 7.8, 8.3 and 8.8 with pH of TB, 8.3 and 8.8 were resolved well. The total running time for the samples to reach the end of the dye front ranged between 2.30 h to 4.30 h at the above pH combinations. It was observed as 3.30 h as total running time under normal pH conditions (RGB, pH 8.8; TB, pH 8.3). At the same time, buffers at higher pH or the combination of extreme pH (7.8 vs. 9.3) in the buffers were not favored good protein mobility/resolution, and bands were diffused. Longer running time was observed in various combinations of pH of RGB and pH of TB with 18.00 h being a longest with a pH of 8.3 and 9.3 for RGB and TB, respectively. This indicated the importance of pH of electrophoresis buffers for ionization of various proteins for better separation.