Electrophoretic concentration of proteins in a nonlinear pH gradient.
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 (+)H3PO4-valine (pI 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 (pI 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.
D006454:Hemoglobins / Q000302:isolation & purification; D006593:Hexokinase / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation; D007526:Isoelectric Point; D007700:Kinetics; D011506:Proteins / Q000302:isolation & purification*; D012260:Ribonucleases / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D006454:Hemoglobins; D011506:Proteins; D012710:Serum Albumin, Bovine; D006593:Hexokinase; D012260:Ribonucleases