Isolation of proteins comprising native gene-specific messenger ribonucleoprotein particles using paramagnetic beads

A method for the rapid isolation of proteins comprising native messenger ri bonucleoprotein particles (mRNPs), that utilises streptavidin-coated parama gnetic beads, coupled with biotinylated oligodeoxyribonucleotides. to pull down general and transcript-specific mRNP complexes from crude cell-free ex tracts. is described. Two biotinylated oligo probes were tested for their e fficacy in isolating mRNPs from tobacco pollen extracts, a 25-mer oligo(dT) probe for the isolation of total mRNPs. and a transcript-specific oligo co mplementary to the pollen-specific ntp303 mRNA sequence. Following annealin g to the target mRNAs. the oligo probes, with associated ribonucleoprotein particles, were captured by streptavidin-coated paramagnetic particles and pelleted in a magnetic field. After several washing steps. the bound RNPs w ere released and analysed by SDS-polyacrylamide gel electrophoresis (SDS-PA GE) and Western blot immunodetection. This one-step mRNP enrichment method could be used either to enhance the detection of low-abundance mRNA-binding proteins in subsequent northwestern and RNA UV cross-linking experiments o r the direct identification and biochemical characterisation of RNA-binding proteins. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.