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A new procedure is described for the isolation of undegraded free and membrane‐bound polysomal mRNA from rat brain in which polysomes are simultaneously separated from soluble components of the cell and slowly sedimenting ribosome species and concentrated by rate‐zonal centrifugation on short linear sucrose gradients. This avoids the problem encountered in conventional procedures of pelleting polysomes along with membranous materials that are not solubilized by detergents and that appear to contain traces of nucleases. It also permits a more thorough analysis of the mRNA populations actively engaged in protein synthesis, since both polyadenylated and nonpolyadenylated mRNAs are isolated together. Moreover, the likelihood of sedimenting nonpolysomal mRNP particles along with polysomes is reduced by using rate‐zonal rather than pelleting centrifugation. The translational activity in vitro of free and membrane‐bound polysomal RNA prepared in this way is high and is about 1.5 times that of RNA prepared by a conventional pelleting technique. The difference is attributable to better preservation of large mRNAs, as inferred from two‐dimensional gel electrophoretic analysis of translation product abundance. The recovery of both classes of polysomal RNA is about 90%. The method is simple, efficient, and adapted for isolation of small amounts of polysomal RNA.

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A simple method is described for enhancing the resolution and detectability of translation products in two-dimensional mapping of reticulocyte lysate translation systems. Assays are fractionated by affinity chromatography on human haptoglobin-Affi-Gel 15 to remove hemoglobin and translation products are eluted with sodium dodecyl sulfate, buffered at pH 9.2. Translation products purified in this. way have less than 10 % of the hemoglobin present in the original assay. The recovery of translation products is about 95 % after haptoglobin chromatography and about 75 % after concentration for mapping. Hemoglobin removal not only enhances the resolution and detectability of low and moderate abundance translation products but also reduces horizontal and vertical streaking in two-dimensional mapping without significantly altering the distribution of translation products.