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

Abstract

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.

PSF and p54nrb/NonO – multi‐functional nuclear proteins

“…This family of proteins is highly homologous in the two C‐terminal RRMs, implying a role in RNA processing. Yet, prior to their cloning, PSF and p54 nrb /NonO were characterized as a p100/p52 DNA‐binding heterodimer [14] and indeed these proteins bind to dsDNA, ssDNA and RNA [2,8,15,16]. Several groups have identified PSF and p54 nrb /NonO in a variety of systems and complexes.…”

Strategies for the enrichment and identification of basic proteins in proteome projects

“…9.0) are rarely separated using 2-DE. In recent times, novel IPGs have become available for separating basic proteins within the pH ranges 6-11 [14][15][16][17], 7-10, 8-11 [18] and 9-12 [1,9]. Alkaline proteins are technically challenging for 2-DE for several reasons.…”

Contributions of mass spectrometry in the study of nucleic acid‐binding proteins and of nucleic acid–protein interactions

“…One such application was described in Guillonneau et al (2001a) and Guillonneau et al (2001b). Figure 4, reproduced from Guillonneau et al (2001b), illustrates what we describe below.…”

“…One such application was described in Guillonneau et al (2001a) and Guillonneau et al (2001b). Figure 4, reproduced from Guillonneau et al (2001b), illustrates what we describe below. Indeed, in these works the proteins contained in crude HeLa nuclear extracts (see Dignam, Lebovitz, & Roeder, 1983 for a detailed preparation protocol) were initially separated in a first dimension with an optimized isoelectric‐focusing electrophoresis (this step separates the proteins according to their isoelectric pH; the migration medium was a gel strip with an immobilized pH gradient).…”

“…The autoradiography shown on the panel A clearly indicates that the binding of the radio‐labeled nucleic acid probe to membrane‐bound proteins is specific, because only clearly defined spots are detected. The matching between the two images allows the excision from the gel of the located proteins for their subsequent proteolysis and analysis by MS. (From Guillonneau et al, 2001b, with permission Copyright 2001 Wiley‐VCH.) [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]…”