Biology
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在肾脏皮质提取物中演示血管内皮生长因子与叶黄素激素之间的线性关系
Chapters
Summary January 22nd, 2020
Please note that all translations are automatically generated.
Click here for the English version.
这里介绍的是利用皮质肾提取物制备和总蛋白质正常化，以证明血管内皮生长因子和哺乳动物肾脏叶黄素激素之间的相关性的协议。
Transcript
我们的协议指出，使用牛和猪的肾脏提取物是研究肾脏生理学的一个很好的经济资源，特别是研究血管内皮生长因子与其他分析物之间的相关性。将VEGF（器官血管生成中一种重要的生长因子）与其他蛋白质关联始终是一个挑战。这种技术使我们能够检查VEGF与激素等分析物之间的关联。
这肯定会促进我们关于这一重要增长因素的知识基础。除了本视频中提及的外，此方法可能有助于将肾皮质提取物中的其他分析物关联。可视化演示将帮助其他人在不出现任何采样错误的情况下获得可重现的结果。
使用新鲜组织对于这项技术的成功至关重要。从动物屠宰后立即获得新鲜的整个肾脏是重要的。总是将组织转移到冰上实验室。
使用无菌剪刀、钳子、刀和培养皿解剖肾脏并切除所需的组织部分。沿着下垂平面轻轻地将肾脏切成两半，然后从肾脏中心的皮层区域切下一块组织。用刀将组织切成小块，用一毫升冰冷1X RIPA裂解萃取缓冲液将其转移到微烟管中。
用特定的样品细节标记管子，并放在冰上。使用带无菌探针的手持均质器使组织均质一到两分钟，直到看不到块，然后在 4 摄氏度下立即将管在 9，600 次 g 下离心，5 分钟。离心后，为ELISA和总蛋白分析准备样品的单独等分，以避免多个冻结/解冻周期。
在开始检测之前，将所有试剂和检测板带到室温下，去除多余的孔，并将其储存在四摄氏度，直到进一步使用。用双蒸馏水将 20X 洗涤缓冲液稀释至 300 毫升，并在没有任何溶液的情况下设置空白井。在每个井中加入50微升的标准或样品，再加50微升的马萝卜过氧化物酶结合，然后立即在每个井中加入50微升的抗体溶液并密封板。
混合板，在37摄氏度下孵育一小时。孵育后，用200微升的洗涤缓冲液清洗每井。在每个孔中加入50微升基板 A 和基板 B，通过敲击侧面轻轻混合板，然后在黑暗中密封并孵育 37 摄氏度，孵育 15 分钟。
在每个井中加入 50 微升的止损溶液。轻拍板，使用分光光度计读取 450 纳米的吸光度。准备试剂和洗涤缓冲液，如前所述，然后在每个井中加入100微升的标准或样品，密封板，混合良好，并在37摄氏度下孵育两小时。
去除每个井中的液体，加入100微升检测试剂 A.密封板，再孵育一小时。接下来，用400微升的洗涤缓冲液清洗每一个井，然后向每井添加90微升基板溶液，轻轻敲击板，并在37摄氏度下再孵育一小时。最后一次孵育后，在每井中加入50微升的止动液，轻轻敲击板，用分光光度计读取450纳米的吸光度。
使用标准的 BSA 测定估计牛和猪肾提取物的总蛋白，并使用此估计值使LH和VEGF-A-ELISA结果正常化。对动物和性别计算了LH和VEGF的均值和中位数。在验证数据正态性后，利用线性回归模型来研究LH与VEGF之间的关系。
LH在牛和猪肾脏中均有VEGF的强而显著的预测。尝试此过程时，必须记住在每个组织中采样相同的区域。类似的程序可用于提取任何类型的组织。
请记住，使用您使用的任何组织的总蛋白提取物使分析物正常化。
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