Development of a novel ampholyte buffer for isoelectric focusing: electric charge-separation of protein samples for X-ray crystallography using free-flow isoelectric focusing.

The purification of biological macromolecules has remained a severe bottleneck in three-dimensional structure determination in the field of protein crystallography. By the use of the Free Flow Electrophoresis (ProTeam FFE; TECAN Group Ltd) apparatus, target protein samples can be purified in a high yield, while a preparative separation is performed in solution without using solid matrices such as polyacrylamide gel. A novel ampholyte buffer suitable for crystallization in the use of the ProTeam FFE apparatus has been developed. This buffer is able to generate pH gradients owing to the use of low-molecular-weight electrolytes. The effect of the flow rate on the pH gradient using the novel ampholyte buffer has been elucidated. When the flow rate was lowered to 78 ml h(-1), the pH gradient in the electrophoretic chambers was generated in an almost linear fashion.