Hopiavuori Blake R, Masser Dustin R, Wilkerson Joseph L, Brush Richard S, Mandal Nawajes A, Anderson Robert E, Freeman Willard M
Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
Department of Ophthalmology, Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd., Oklahoma City, OK, 73104, USA.
Methods Mol Biol. 2017;1609:33-41. doi: 10.1007/978-1-4939-6996-8_4.
Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (Nuclear), P2 (Cytoskeletal), P3 (Neurosynaptosomal), PSD (Post-synaptic Densities), and SV (Synaptic Vesicle).
蔗糖梯度离心是一种非常有用的技术，可根据特定膜的大小和密度来分离它们。这对于检测靶向特定膜的脂肪酸和脂质分子特别有用。如果不进行分级分离，这些类型的分子在被更丰富且分布在各种细胞膜中更为普遍的脂质分子稀释后，可能会低于检测水平。分离这些脂质集中的特定膜类型有助于对其进行检测和分析。我们在此描述我们的突触膜分离方案，该方案从起始神经组织匀浆中可出色地分离出五个主要膜组分，且分辨率清晰：P1（核膜）、P2（细胞骨架膜）、P3（神经突触体膜）、PSD（突触后致密物膜）和SV（突触小泡膜）。
