ISOLATION OF HUMAN SERUM TRANSFERRIN BY FREE-FLUID RECYCLING ELECTROPHORESIS IN SIMPLE BUFFERS

The isolation of human serum transferrin (Tf) using recycling isotachophoresis (RITP) and recycling isoelectric focusing (RIEF) with simple buffers is described. Serum fractionation, the first step in the protocol for Tf purification, is shown to be easily performed either by RITP of filtered serum using low molecular mass spacers or by RIEF of dialyzed serum employing a binary mixture of a well-defined buffer pair covering the pH range between 5.2 and 6.2, called RIEF-OptiFocus. For polishing, Tf-containing fractions are reprocessed by RITP or RIEF-OptiFocus. Other RIEF approaches based on the use of single amino acids at high concentration and ternary amino acid mixtures are shown to constitute less effective methods for Tf isolation. With a four-step protocol, comprising in turn RITP, RIEF-OptiFocus, ultrafiltration and again RITP, ''single-band'' purity (as assessed by two-dimensional gel electrophoresis) is obtained. Omission of the first step (RITP) and direct processing of dialyzed serum by RIEF-OptiFocus, ultrafiltration and RITP, is shown to provide remarkable results as well.