Electrophoretic transfer of proteins across polyacrylamide membranes

The electrophoretic transfer of purified proteins has been examined in a Gr adiflow "Babyflow BF100" unit. A number of factors affect protein separatio n within this preparative electrophoresis system. We established that the r ate of protein transfer was proportional to the applied voltage. The transf er is slowest at the isoelectric point (pI) and increased the further away the pH was from the pI of the protein. Protein transfer was found to be ind ependent of the ionic strength of the buffer, for buffers that excluded the addition of strong acids or strong bases or sodium chloride. Transfer decr eased as the pore size of the membrane decreased. Finally, transfer was inh ibited at high salt concentrations in the protein solution, but remained un affected when urea and non-ionic detergents were added to the solution. To increase the speed of protein separations, buffers with low conductivity sh ould be used. A pH for the optimal separation should be selected on the bas is of the relative pI and size of the target proteins and that of the major contaminants. (C) 1999 Elsevier Science B.V. All rights reserved.