AssayGenie高灵敏度脂肪酸氧化 (FAO) 检测试剂盒的应用
脂肪酸每碳提供的ATP比葡萄糖等碳水化合物多。 在具有高能量需求的组织中,例如心脏组织,高达 50 – 70%的能量来自脂肪酸β-氧化,可转化脂肪 酸制乙酰辅酶A,同时产生FADH2和NADH 线粒体。
这种严格的有氧氧化途径由四个不同的步骤组成: 酰基辅酶A脱氢酶脱氢,烯酰辅酶A水合 水合酶,通过 3-羟基酰辅酶 A 脱氢酶和硫解酶脱氢 β-酮硫醇酶裂解。
研究脂肪酸氧化的重要性
β-氧化系统在人体中的重要性体现在 存在一组被归类为脂肪酸氧化的遗传疾病 疾病(FAOD),这是一组异质性脂肪酸缺陷 转运和氧化,并遗传为常染色体隐性遗传病, 具有广泛的临床表现。
此外,有证据表明,粮农组织在粮农组织活动期间的监管 胎儿发育不仅对胎儿生命很重要,而且 对成年期健康和疾病的影响。粮农组织的衡量标准 因此,活动为病理生理学和病理学提供了有价值的见解 许多疾病的代谢基础。
检测精灵非放射性 粮农组织的测定基于底物辛酰辅酶A的氧化。生成 NADH与四唑盐INT还原为甲胺素偶联。 红色甲臜的强度与粮农组织的增加成正比 活动。测定溶液和底物应等分储存在 -80°C.
Assay Genie检测精灵脂肪酸氧化检测试剂盒是一种高度灵敏的试剂盒 用于定量测量脂肪酸的比色法 细胞和组织中的氧化。
这种检测方法利用 高可溶性辛酰辅酶A底物,可实现 脂肪酸氧化与长链低溶性的测量 底物如18C不饱和脂肪酸油酸酯。
上图:脂肪酸氧化测定:组织 裂解物由健康的仓鼠心脏和 与心力衰竭相关的TO2仓鼠模型。裂解物 用于测定和脂肪酸氧化水平 量化。
下图:脂肪酸氧化测定: 辛酰辅酶A是底物,而不是反应的产物。请 见图。
细胞/组织提取物的制备:
1.用冰冷的磷酸盐缓冲盐水(PBS)洗涤~106细胞两次, 并从细胞沉淀中去除PBS。细胞沉淀应 储存在-80ºC.组织样品应用PBS洗涤至 去除血细胞,这可能导致检测结果不一致。
2.通过稀释 1x 细胞裂解液制备足够的 10x 细胞裂解液 用冰冷的dH 2 O溶液。加入 50 – 100 μl 冰冷的 1x 细胞裂解 细胞沉淀溶液。通过上下移液提取细胞 (温和)。将裂解物留在冰上5分钟 间歇性轻微激动。如果裂解物是粘稠的,添加更多的1x细胞 裂解溶液并重复移液。在 以最大速度冷藏微量离心机5分钟。回收上清液 用于测定。将组织在 1x 细胞裂解液中匀浆,并裂解 通过离心澄清。每 25.0 毫升 5x 使用 ~1 毫克组织 用于组织匀浆的细胞裂解溶液。
3.进行蛋白质测定以确定样品蛋白质浓度。 通过用冰冷的 1x 稀释来标准化样品蛋白质浓度 细胞裂解液至1 – 3毫克/毫升。将蛋白质样品保存在冰上 次。冻融的粗蛋白裂解物可表现出酶还原 活动。裂解物应储存在-80ºC。 注意:不要使用缓冲液 含有还原剂或SDS。
试剂解冻:
将解冻的粮农组织化验溶液和20倍粮农组织底物放在冰上。轻轻搅拌 移液前的溶液。重要的是要尽量减少时间 试剂解冻。使用后立即冷冻溶液。
对照溶液和反应溶液的制备:
1.对照溶液由1份dH 2 O和20份混合制备 粮农组织化验解决方案,例如25μldH 2 O与500μlFAO化验混合 溶液。测定过程中将新鲜制备的对照溶液保持在冰上。
2.反应溶液通过混合1份20x FAO底物和 20份粮农组织化验溶液,例如25μl 20x粮农组织底物与 500μl粮农组织测定溶液。将溶液放在冰上并立即使用。 每个蛋白质样品用50μl对照溶液和50μl处理 反应溶液分为两组。
Assay Genie检测精灵脂肪酸氧化检测试剂盒的应用
研究健康和病理状态(脂质代谢)的代谢途径和过程。
信号转导途径的研究。
遗传性疾病的研究,例如脂肪酸氧化障碍(FAODs)*。
心血管疾病的研究,例如心力衰竭。
胎儿发育过程中细胞能量代谢的研究。
脂肪酸氧化在癌症代谢中的影响。
*此试剂盒仅供研究使用,不能用于诊断 人类疾病。
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