IMPROVED AND SIMPLIFIED IN-GEL SAMPLE APPLICATION USING RESWELLING OFDRY IMMOBILIZED PH GRADIENTS

A simple and inexpensive methacrylate rehydration chamber was built to accomodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein ent ering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves wi th the gel In contact with 500 mu L of sample for 6 h or overnight. Th is avoided the use of sample cups, eliminated precipitation al the sam ple application site, thus improving resolution over the entire pH ran ge of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of re agents during rehydration and equilibration owing to the reduced volum es. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.