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A Rapid Extraction Method for mammalian cell cultures suitable for quantitative immunoblotting analysis of proteins including phosphorylated GCN2 and eIF2α

机译：一种适用于哺乳动物细胞培养的快速提取方法适用于蛋白质的定量免疫印迹分析包括磷酸化的GCN2和eIF2α

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class="kwd-title">Method name: Rapid Protein Extraction Method (RPE method) class="kwd-title">Abbreviations: eIF2α, α subunit of translation initiation factor 2, GCN2, General Control non-derepressible 2, PBS, phosphate-buffered saline, SDS-PAGE, SDS polyacrylamide gel electrophoresis class="kwd-title">Keywords: Mammalian cell culture, cell lysis, SDS-PAGE, quantitation, western blot, GCN2, eIF2 alpha class="head no_bottom_margin" id="abs0010title">AbstractMany studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are: class="first-line-outdent" id="lis0005">
  • • No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors.
  • • The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary.
  • • The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method.
    • 机译：<！-fig ft0-> <！-fig @ position =“ anchor” mode =文章f4-> <！-fig mode =“ anchred” f5-> <！-fig / graphic | fig / alternatives / graphic mode =“ anchored” m1-> class =“ kwd-title”>方法名称：快速蛋白质提取方法（RPE方法） class =“ kwd-title”>缩写： eIF2α，翻译起始因子2的α亚基，GCN2，通用控制不可抑制2，PBS，磷酸盐缓冲液，SDS-PAGE，SDS聚丙烯酰胺凝胶电泳 class =“ kwd-title”>关键字： 哺乳动物细胞培养，细胞裂解，SDS-PAGE，定量，蛋白质印迹，GCN2，eIF2 alpha class =“ head no_bottom_margin” id =“ abs0010title”>摘要许多研究都需要检测和相对使用免疫印迹从细胞培养样品中定量蛋白质。限制因素是蛋白酶抑制剂的成本，破坏细胞和产生样品所需的时间以及细胞破坏过程中蛋白质损失的高风险。另外，一个常见的问题是由于释放的基因组DNA而导致裂解样品的粘度升高。结果，在变性聚丙烯酰胺蛋白凝胶电泳（SDS-PAGE）之前，例如，需要先分解DNA。使样品通过注射器针头，超声或DNase处理。为了寻找一种更具成本效益，快速且稳健的方法，我们发现细胞裂解，蛋白质变性和DNA片段化仅需两个步骤即可完成：收获，然后进行简单的非繁琐的第二步。与许多先前存在的细胞破碎程序类似，在我们的快速蛋白质提取（RPE）方法中，从细胞中释放的蛋白质会立即暴露于变性环境中。但是，我们方法的优点是： class =“ first-line-outdent” id =“ lis0005”> <！-list-behavior =简单前缀-word = mark-type = none最大标签大小= 9 ->
  • •不需要破坏缓冲液，而是将蛋白质直接释放到用于SDS-PAGE的变性蛋白质上样缓冲液中。因此，我们的RPE方法不需要任何昂贵的抑制剂。
  • •RPE方法不涉及裂解后离心步骤；取而代之的是，在第2步（此方法新的混合热处理步骤）中将所有细胞材料溶解。这样可以防止在离心过程中可能发生的蛋白质损失。此外，第二步同时剪切了基因组DNA，因此无需进行DNA片段化的额外步骤。
  • •生成的样品适用于高质量的定量免疫印迹。通过我们的RPE方法，我们成功地定量了蛋白激酶GCN2及其底物eIF2α的磷酸化形式。实际上，与使用现有方法生成的样本相比，西方信号更强且背景更少。

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