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.
Guillonneau
F
F
INSERM U201, CNRS UMR 8646, Muséum National d'Histoire Naturelle, Paris, France.
Labas
V
V
Auvin
C
C
Praseuth
D
D
D004268:DNA-Binding Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D013058:Mass Spectrometry; D016601:RNA-Binding Proteins / Q000032:analysis*; D015203:Reproducibility of Results
D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins