Recycling isoelectric focusing: use of simple buffers

Using the recycling free-flow focusing (RF3) apparatus, we have demonstrated that single ampholytes can be utilized to establish very stable pH regions, separating all proteins into three groups: a sharply resolved zone of proteins isoelectric at the prevailing pH, this "pH window" being bracketed by zones of more acidic and/or basic proteins. The ampholytes used are either amino acids or their dipeptides and other derivatives. Where necessary, because of lack of an ampholyte with the required pH, a binary mixture of ampholytes can be utilized. The closer their isoelectric points (pI), the narrower will be the pH window, i.e., the sharper the resolution of the bracketed proteins. This method overcomes the necessity of using ill-defined commercial carrier ampholytes, such as Ampholine, for preparative isoelectric focusing. It is recommended that the ampholytes be utilized at relatively high concentration, 100mM or higher, this contributing to pH stability and minimizing protein precipitation.