Kopec Ashley M, Rivera Phillip D, Lacagnina Michael J, Hanamsagar Richa, Bilbo Staci D
Department of Psychology & Neuroscience, Duke University, Durham, NC, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
Department of Psychology & Neuroscience, Duke University, Durham, NC, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
J Neurosci Methods. 2017 Mar 15;280:64-76. doi: 10.1016/j.jneumeth.2017.02.002. Epub 2017 Feb 13.
Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets.
We optimized a buffer for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer.
Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples.
COMPARISON WITH EXISTING METHOD(S): Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation.
This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects.
同时评估多个分子加工水平的技术很有吸引力，因为复杂神经现象背后的分子信号传导发生在互补水平。TRIzol法可分离RNA和DNA，但由于沉淀的蛋白质沉淀溶解效率低，蛋白质提取困难。
我们优化了一种缓冲液，用于从TRIzol沉淀的脑组织中高效溶解蛋白质以进行蛋白质印迹分析，该缓冲液在直接匀浆脑组织方面也比RIPA缓冲液更有效。
通过优化标准裂解缓冲液中化学物质的浓度，不仅溶解过程中的蛋白质产量增加，直接匀浆后的蛋白质产量也增加。总蛋白质产量和翻译后修饰分析的有效孵育参数非常灵活。重要的是，溶解的蛋白质样品中代表了不同的神经细胞类型和蛋白质类别。此外，我们使用解离的小鼠脑组织从其他细胞类型中分离小胶质细胞，并成功从这些难以获得的小样本中解析出细胞类型特异性蛋白质。
迄今为止，溶解缓冲液主要由SDS或尿素组成；本文数据表明，裂解缓冲液中的常见成分在直接匀浆后和沉淀后也能增强蛋白质溶解。
该方法适用于评估来自单个脑样本的基因和蛋白质表达，在尽量减少实验对象数量的同时，更全面地评估神经现象。
