Capillary Isoelectric Focusing (CIEF)
Capillary isoelectric focusing (CIEF) is a "high resolution" electrophoretic technique used to separate peptides and proteins on the basis of pI. CIEF has been used successfully to measure protein pI and for the separation of protein isoforms, and other proteins, e.g. immunoglobulins and hemoglobins. As with SDS-protein, this is a well-established gel electrophoretic technique adapted to the CE format.
In CIEF a pH gradient is formed within the capillary using ampholytes. Ampholytes are molecules that contain both an acidic and a basic moiety (that is, they are zwitterionic) and can have pI values that span the desired pH range of the CIEF experiment (pH 3-10, for example). After filling the capillary with a mixture of solute and ampholytes, the gradient is formed upon applying a voltage. With a basic solution at the cathode and an acidic solution at the anode, upon application of the electric field the charged ampholytes and proteins migrate through the medium until they reach a region where they become uncharged (at their pI). This process is know as "focusing". The protein zones remain narrow since a protein which enters a zone of different pH will become charged and migrate back.
The status of the focusing process is indicated by the current. Once complete, a steady-state is reached and current is minimal. After focusing, the solutes and ampholytes are mobilized and the zones passed through the detector. Mobilization can be accomplished by either application of pressure to the capillary or by addition of salt to one of the reservoirs.
EOF needs to be reduced or eliminated in CIEF since the flow could flush the ampholytes from the capillary before focusing is complete. Reduction of EOF can be accomplished by the use of dynamic or covalent coatings. The dynamic coatings have the advantage of simplicity, but obtaining reproducible EOF is often difficult. The coating, either dynamic or covalent is also helpful in limiting protein adsorption to the capillary walls.
Since the solute is loaded onto the capillary during filling with the amphoteric solution, it is possible to load significantly larger volumes than in most other CE modes. Precipitation, which results from very high protein concentrations within the zones, is usually the limiting factor in sample loading. |