Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada.
Department of Mathematics and Statistics, McGill University, Montreal, Quebec, Canada.
Electrophoresis. 2022 Oct;43(20):2014-2022. doi: 10.1002/elps.202100364. Epub 2022 Sep 4.
Inorganic polyphosphates (polyPs) have been identified in eukaryotic and prokaryotic cells alike. Various extraction methods have been optimized as a necessary step before identification and measurement of these polymers. Three commercially available sodium polyP glasses were either dissolved or dissolved and extracted by two commonly used polyP extraction techniques - perchloric acid or buffered phenol-chloroform. The products were separated by polyacrylamide gel electrophoresis (PAGE), stained with toluidine blue O, and the migration results quantitatively compared. Both extraction processes reduced the relative migration distances of the peak and leading edges, and the stained band lengths, suggesting reduced polyP migration and dispersion. P diffusion-ordered spectroscopy nuclear magnetic resonance confirmed that polyP extraction by perchloric acid or phenol-chloroform processes reduced polyP diffusion coefficients and suggested hydrolytic degradation with stronger end-chain signals. Reduced polyP diffusivity after extraction makes possible an overestimation of synthetic polyP chain length assignment when compared to unextracted polyP ladders with PAGE. The mechanism(s) for reduced synthetic polyP diffusion after extraction and intracellular chemical environment effects on migration are not known.
无机多聚磷酸盐（polyPs）在真核和原核细胞中都有被发现。在鉴定和测量这些聚合物之前，已经优化了各种提取方法作为必要步骤。三种市售的多聚磷酸钠玻璃，无论是溶解还是溶解后，都可以通过两种常用的多聚磷酸提取技术——高氯酸或缓冲酚-氯仿进行提取。产物通过聚丙烯酰胺凝胶电泳（PAGE）进行分离，用甲苯胺蓝 O 染色，并定量比较迁移结果。这两种提取过程都减少了峰和前缘的相对迁移距离以及染色带的长度，表明多聚磷酸盐的迁移和分散减少。扩散排序波谱核磁共振证实，高氯酸或酚-氯仿提取过程减少了多聚磷酸盐的扩散系数，并提示具有更强末端链信号的水解降解。提取后多聚磷酸盐扩散性降低，与未经提取的 PAGE 多聚磷酸盐梯相比，可能会高估合成多聚磷酸盐链长的分配。提取后合成多聚磷酸盐扩散性降低的机制以及细胞内化学环境对迁移的影响尚不清楚。
