High resolution two-dimensional polyacrylamide gel electrophoresis using Immobilines. Application to the study of...

Cytoplasmic soluble proteins of unknown origin but which could be associated with the development and maturation of the central nervous system are recognized by antibodies found in serum of patients with a paraneoplastic neurological syndrome. The associated antigens are of great interest in understanding these neurological pathologies. To study these antigens we describe and criticize a two dimensional polyacrylamide gel electrophoresis (2D-PAGE), using immobilized pH gradient (IPG) in first dimension. Proteins from brain complex mixtures were separated by this technique in order to identify and characterize (molecular weight, p-isoelectric point, sequencing) a protein of interest. Results prove the high reproducibility and the good resolution of the technique. Without applying enrichment method prior to 2D-PAGE, any proteins are not sequenceable after transblotting to PVDF (polyvinylidene difluoride) membrane and Coomassie blue staining. In these cases the meaning to reach the wanted aim, i.e. sequencing, was discussed.