Post-trizol protein extraction from peripheral blood mononuclear cells

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