Electrophoretic concentration of proteins in a nonlinear pH gradient

Abstract A method for electrophoretic concentration of differently charged proteins is described. A nonlinear pH gradient is generated by imposing a potential gradient on an electrolyte system composed of (+)H 3 PO 4 -valine (p I 6.0)-Servalyte (pH 9–11)-triethylamine(−). Proteins contained in the valine solution accumulate at the interphase formed between the valine solution and the Servalyte solution. This interphase acts as a barrier or liquid membrane to all proteins having isoelectric points in the range 6–9. For proteins having isoelectric points in the range 5–7 valine is replaced by histidine (p I 7.64) and the Servalyte by Pharmalyte, pH 2.5–5.0. Ribonuclease, hexokinase, bovine serum albumin, and hemoglobin were concentrated and recovered from the top of the column using a peristaltic pump. The duration of concentration process was 1–4 h, the length of the run depending on the experiment scale (20 or 100 ml protein solution), the amount of protein, and the isoelectric point of the protein. Proteins were concentrated 9- to 48-fold, depending on the initial volume and concentration of the protein. The recoveries ranged from 79.7 ± 1.1 for hemoglobin to 93.17 ± 2.84 for ribonuclease.