Post-TRIzol protein extraction from peripheral blood mononuclear cells Scientific paper

Abstract

After sample processing for RNA and DNA analysis, the leftover protein pellets are usually discarded due to the limited efficiency of pellet reconstitution/solubilisation. As the pelleted proteins are tightly packed, they are most often solubilised using chaotropic agents (e.g., guanidine hydro­chlo­ride or urea), detergents (e.g., SDS), salts (NaCl) or basic buffer (Tris). The aim of this study was to define and optimise the procedure for the efficient extraction of proteins from human peripheral blood mononuclear cells (PBMCs), obtained by a single blood draw and lysed in TRIzol reagent, by varying experimental conditions in terms of protein precipitation solvent (iso­propanol or acetone), washing (with or without guanidine hydrochloride) and solubilisation solution (containing SDS, NaCl, urea and/or Tris). We evaluated the efficacy of the final, optimised protocol to solubilise both small cytoplas­mic and larger transmembrane proteins, and the compatibility with methods employed for the subsequent analysis of protein posttranslational modific­ations, such as glycosylation. The optimised protocol for the extraction and iso­lation of post-TRIzol leftover proteins from PBMCs can be defined as fol­lows: protein precipitation from the organic phase with ice-cold acetone, pellet wash­ing with absolute ethanol and solubilisation in 1 % SDS, employing 20 min heating at 50 °C and vortexing.