A new technique for desorbing substances tightly bound to affinity gels: flat-bed electrophoretic desorption in Sephadex via isoelectric focusing (FEDS-IEF)

In some cases, proteins and other molecules which are tightly bound to affinity gels can be recovered under mild conditions by electrophoresis. We have extended this technique by running electrophoretic desorption in flat-beds of Sephadex in the presence of ampholytes (FEDS-IEF). A number of advantages of this technique are noted: due to the geometry of the apparatus, high voltages can be used which result in short running times; there are no physical barriers to the migration of the protein and no abrupt conductivity drops; desorbed samples are easily located and recovered; and relatively large sample loads can be readily accommodated. Running times are very sensitive to the experimental conditions. Affinity gels should be applied as a narrow zone, distant from the anticipated banding position of the desorbed species. A wide ampholyte interval is generally recommended. The system appears to be gentle and flexible enough to allow investigators to optimize the conditions for desorption of various affinity gel systems.