A reliable and simple method for two-dimensional electrophoresis and identification of HeLa nuclear alkaline nucleic acid-binding proteins using immobilized pH gradient.

Alkaline proteins were separated by two-dimensional electrophoresis, using isoelectric focusing in commercial pH 6-11 immobilized pH gradients (IPG), in order to identify nucleic acid-binding proteins by South- or Northwestern blotting. The corresponding spots were chosen according to their DNA or RNA binding properties, excised, and submitted to a simplified tryptic digestion and peptide extraction protocols. Matrix assisted laser desorption/lonization-time of flight (MALDI-TOF)-mass spectrometry was used to identify 36 out of 39 excised protein spots. The database search output gave a set of proteins already known as DNA or RNA binding factors, some of which have enzymatic activity (RNA-processing, splicing, cleavage, homologous DNA recognition, transcription factor). The method can be performed entirely using commercially available products, from HeLa nuclear extracts to IPG-gradients.