Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium: A methodological approach to improve target protein quantification.
Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium: A methodological approach to improve target protein quantification.
复制标题
培养基中血清白蛋白对原代神经元培养物中蛋白质分析的失真：改进靶蛋白定量的方法学方法。
DOI：
10.1016/j.jneumeth.2018.07.002
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发表时间：
2018
影响因子：
3
通讯作者：
Willis A
中科院分区：
文献类型：
--
作者：
Willis A
背景原代神经元培养是各种神经科学实验的基础，包括各种蛋白质分析技术，如Western blotting，需要从培养的神经元中提取蛋白质。在免疫印迹实验中，我们遇到了一些问题，因为细胞提取物中存在65-70 KDa的高度丰富的蛋白质，这干扰了总蛋白质的估计，以及对类似大小的目标蛋白质的免疫检测。以前的研究表明，血清蛋白，特别是白蛋白，包含在常用的培养液中，可以与普通细胞培养塑料器皿结合或被吸附。新方法我们对传统细胞裂解/提取方法的洗涤步骤进行了简单的修改。结果我们报道了从标准培养基中积累的大量白蛋白与细胞内容物一起从原代神经元培养中提取出来。与现有方法相比，从神经元培养液中累积的白蛋白的数量相当于细胞含量，可能会扭曲来自总蛋白分析的数据和来自附近条带的免疫反应信号。通过改变蛋白质提取过程中的洗涤程序，这些问题可以得到改善。结论我们认为，原代神经元培养的标准延长的培养周期，加上连续更换培养液的要求，可能会使它们特别容易受到所用培养液中白蛋白的累积污染。最后，我们建议在蛋白质提取方案中实现简单的洗涤步骤，以改善这种干扰。
BackgroundPrimary neuronal cultures underpin diverse neuroscience experiments, including various protein analysis techniques, such as Western blotting, whereby protein extraction from cultured neurons is required. During immunoblotting experiments, we encountered problems due to a highly-abundant protein of 65–70 KDa present in the cell extracts, that interfered with total protein estimation, and immunodetection of target proteins of similar size. Previous research has suggested that serum proteins, specifically albumin, contained within commonly-used culture media, can bind to, or be adsorbed by, generic cell culture plasticware. This residual albumin may then be extracted along with cell proteins.New methodWe made simple modifications to wash steps of traditional cell lysis/extraction protocols.ResultsWe report that a substantial amount of albumin, accumulated from the standard culture media, is extracted from primary neuronal cultures along with the cellular contents. This contamination can be reduced, without changing the culture conditions, by modifying wash procedures.Comparison with existing methodsAccumulated albumin from neuronal culture media, in amounts equivalent to cellular contents, can distort data from total protein assays and from the immunoreactive signal from nearby bands on Western blots. By altering wash protocols during protein extraction, these problems can be ameliorated.ConclusionsWe suggest that the standard extended culture periods for primary neuronal cultures, coupled with the requirement for successive medium changes, may leave them particularly susceptible to cumulative albumin contamination from the culture media used. Finally, we propose the implementation of simple alterations to wash steps in protein extraction protocols which can ameliorate this interference.