| Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells
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Author:
Date:
2020-09-20
[Abstract] As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the substrate of acetyltransferase GCN5L1 and deacetylase Sirt3. Acetylation levels of LCAD and beta-HAD regulate its enzymes activity and thus affect fatty acid oxidation rate. Moreover, immunoprecipitation is a key assay for the detection of LCAD and beta-HAD acetylation levels. Here we describe a protocol of immunoprecipitation of acetyl-lysine and western blotting of LCAD and beta-HAD in palmitic acid treated HK-2 cells (human renal tubular epithelial ...
[摘要] [摘要] 甲作为肾脏的主要能量代谢器官之一，肾脏已被证明具有很高的能量需求，并且更倾向于将脂肪酸代谢为主要能量来源。 长链酰基辅酶A脱氢酶（LCAD）和Beta-羟酰基 -CoA脱氢酶（β-HAD），涉及的关键酶脂肪酸氧化，已被确定为乙酰转移酶GCN5L1和脱乙酰酶Sirt3的底物。 LCAD和β-HAD的乙酰化水平调节其酶的活性，从而影响脂肪酸的氧化速率。 此外，免疫沉淀是检测LCAD和β-HAD乙酰化水平的关键方法。在这里，我们描述了在棕榈酸处理的HK-2细胞（人肾小管上皮细胞）中乙酰赖氨酸的免疫沉淀以及LCAD和β-HAD的免疫印迹实验。 该方案为读者提供了清晰的步骤，因此该方法可用于检测各种蛋白质的乙酰化水平。
[背景 ] 翻译后修饰（PT Ms）使细胞具有高度动态的机制来调节细胞途径（Zhao 等，2010）。 乙酰化已成为主要的翻译后蛋白质修饰之一。越来越多的证据小号指示乙酰化对手磷酸化的线粒体调控修改（Henriksen的等人，2012） 。 过线粒体蛋白质的60％被乙酰化，作为声明，这是参与能量代谢例如三羧酸（TCA）循环，氧化磷酸化（OXPHOS），脂肪酸氧化和氨基酸代谢（Hirschey 等人，2010 ; ...
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| Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria
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Author:
Date:
2020-03-05
[Abstract] The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the ...
[摘要] [摘要 ] 细菌中多药耐药性的出现和迅速扩散导致对新型抗菌剂的迫切需求。膜通透性是许多针对革兰氏阴性细菌的抗菌分子的机制。因此，为了确定潜在的抗菌分子的功效，重要的是评估治疗后细菌膜通透性的变化。这项研究描述了使用荧光探针N-苯基-1-萘胺和碘化丙啶测定外膜和内膜通透性的方案。与其他实验（例如电子显微镜和最小杀菌浓度的测定）相比，该方法提供了一种更简单，更快速且经济高效的方法来评估抗菌分子的膜通透性和杀菌功效。这项研究提出了一种相对于传统方案的优化方案，该方案是在与抗菌测定相同的培养条件下，将细菌与抗菌分子一起孵育，然后在不含肉汤和抗菌分子的缓冲液中检测荧光探针的信号。此协议可避免的效果养分缺乏对细菌的生理状态和抗菌molecul的干扰ES 朝向的F luorescent探针。因此，该方法可以有效，准确地评估膜的渗透性，并与从其他抗菌测定法（如最小抑菌浓度和时间杀灭曲线测定法）获得的结果相匹配。
[背景 ] ...
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