Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma

Nan YANG; Siwei TAN; Yan PENG; Yuanguo ZHOU; Yalei NING

doi:10.16016/j.1000-5404.201805035

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Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma

Basic Medicine | 更新时间：2026-01-09

- Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma
- Journal of Third Military Medical University Vol. 40, Issue 20, Pages: 1846-1850(2018)
- 作者机构：
  
  400042 重庆，陆军军医大学(第三军医大学)第三附属医院(野战外科研究所)分子生物学中心，国家创伤、烧伤与复合伤重点实验室，陆军军医大学脑科学协同创新中心
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81571888);the Project of Basic and Frontier Research Plan of Chongqing(CSTC2014jcyjA10072)
- DOI：10.16016/j.1000-5404.201805035
  CLC： R329-33;R338.1;R446
- Received：06 May 2018，
  
  Revised：2018-07-29，
  
  Published：30 October 2018
- 稿件说明：
移动端阅览
Nan YANG, Siwei TAN, Yan PENG, et al. Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma[J]. Journal of Third Military Medical University, 2018, 40(20): 1846-1850.
DOI：

Nan YANG, Siwei TAN, Yan PENG, et al. Laser microdissection with automated capillary electrophoresis Western blotting for quantification of phosphorylated protein kinase A in hippocampal neurons of mice with mild brain trauma[J]. Journal of Third Military Medical University, 2018, 40(20): 1846-1850. DOI： 10.16016/j.1000-5404.201805035.

摘要

目的

构建及评价收集特定部位的标记神经元，对这类神经元的微量蛋白水平采用定量的方法，并将此方法初步应用于小鼠颅脑创伤后海马亚区神经元中。

方法

硫堇染色未固定处理的冰冻切片脑组织神经元，激光显微切割收集海马CA1区和DG区神经元，全自动毛细管Western blot检测收集细胞的磷酸化蛋白激酶A(p-PKA)蛋白水平。

结果

全自动毛细管Western blot对低浓度蛋白的检测下限低于传统Western blot 1个数量级，其线性相关系数为98.85%，日内和日间精密度分别为2.50%和4.09%。本方法应用于颅脑创伤小鼠脑组织，发现伤后1 d海马DG区神经元p-PKA显著增高(

0.01)，CA1区无变化。

结论

本方法基于激光显微切割和全自动毛细管Western blot，能够实现精确收集指定神经元并准确定量微量蛋白，成功应用于目标神经元蛋白定量，具有在细胞类型众多且混杂分布的组织中进行细胞和分子水平研究的应用前景。

Abstract

Objective

To establish a quantitative detection system for phosphorylated protein kinase A (p-PKA) in labeled hippocampal neurons of mice following mild brain trauma using laser microdissection combined with automated capillary electrophoresis Western blotting.

Methods

From a mouse model of mild traumatic brain injury

fresh samples of the hippocampal tissues were collected for preparing frozen sections with Thionine staining. Laser microdissection was used for collecting the neurons in the hippocampal CA1 and DG regions and the expression level of p-PKA was detected using fully automated capillary Western blotting.

Results

The lower detection limit of automated capillary electrophoresis Western blotting for low-concentration proteins was lower than that of conventional Western blotting by 1 order of magnitude. This combined detection system had a linear correlation coefficient of 98.85% for p-PKA detection with inter- and intra-day precisions (RSD) of 2.50% and 4.09%

respectively. In the mouse model with traumatic brain injury

the level of p-PKA in the hippocampal DG region increased significantly at 1 day after the injury (

0.01)

but its expression showed no significant changes in the CA1 region.

Conclusions

Laser microdissection combined with automatic capillary Western blotting allows collection of specific neuronal cells and accurate quantification of low-concentration proteins

and can be potentially used for cellular and molecular studies of tissues with complex distribution of different cell types.

关键词

Keywords

references

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