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A reliable and simple method for two‐dimensional electrophoresis and identification of HeLa nuclear alkaline nucleic acid‐binding proteins using immobilized pH gradient
Author(s) -
Guillonneau François,
Labas Valérie,
Auvin Catherine,
Praseuth Danièle
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200112)22:20<4391::aid-elps4391>3.0.co;2-8
Subject(s) - nucleic acid , isoelectric focusing , rna , dna , chemistry , chromatography , electrophoresis , gel electrophoresis , oligonucleotide , microbiology and biotechnology , biochemistry , biology , enzyme , gene
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/Ionization – 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.