Blotting from Immobilized pH Gradient Gels: Application to Total Cell Lysates

Isoelectric focusing as used in the first dimension of two-dimensional gel electrophoresis separates protein isoforms such as those due to phosphorylation and acetylation. The immunoblotting method described here reveals this diversity by a one-dimensional separation. Using commercially available immobilized pH gradient plates or strips, the resolved proteins are transferred to PVDF membranes by diffusion and are probed with protein-specific antibody. The system is useful for monitoring changes of banding patterns and permits parallel processing of samples. Since the effect of posttranslational modifications on the isoelectric point can be predicted, inferring the number and extent of modifications is possible.