The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, front chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2. the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5 h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60-65% from one egg white with minimal lysozyme activity detected. (C) 2002 Elsevier Science (USA). All rights reserved.