Steady‐state stacking in agarose at various pH

The applicability of multiphasic buffer systems to electroendosmosis‐free agarose (IsoGel) was tested in order to be able to combine the benefits of agarose gel electrophoresis with those of steady‐state stacking. It was found that buffer systems of a negative polarity function normally over the entire pH range, whereas proteins with positive net charge are retarded at the gel surface in buffer systems with positive polarity. This retardation is not due to a failure to establish a moving boundary in agarose. Rather, it appears due to a negatively charged weakly acidic functional group in agarose which can be removed from agarose by pre‐electrophoresis at 0.1 M ionic strength. After its removal positively charged proteins also stack normally in multiphasic buffer systems of the positive polarity.