Enrichment of Plasma Membranes from Small Brain Samples by Aqueous Polymer Two-Phase Systems
Plasma membrane (PM) proteins take center-stage in most of the fundamental processes of the nervous system. They impart specificity to the formation of neuronal circuits, and determine the mode of neurotransmission. To accomplish these tasks, they demonstrate spatially and temporally restricted expression and regulation. Consequently, molecular analysis has to focus on functionally or anatomically distinct areas of the nervous system in order to get physiological relevant insights into PM proteomes. This constriction results most often in minute amounts of sample. The low abundance of PM proteins when compared to other subcellular proteomes such as those from mitochondria and cell nuclei further aggravates their analysis. Finally, posttranslational modifications of PM residing proteins differ from those observed in the secretory, endocytic, and recycling pathways. Taken together, all these properties of PM proteins require a protocol, which fosters their selective enrichment from small samples at high yield and high purity. Here, I will present a protocol based on partitioning of subcellular membranes in aqueous polymer two-phase systems, which fulfills these criteria. In contrast to previous protocols, I introduced an adaptation to preserve phosphorylation sites. The protocol was previously shown to provide profound insights into physiological relevant PM proteomes. By doing so, the protocol furthermore represents an excellent starting point towards identification of novel drug targets or key regulatory mechanisms such as phosphorylation.
Key wordsNervous system Plasma membrane Aqueous polymer two-phase system Countercurrent distribution Phosphoproteome
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