Use of the REVERT(®) total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states.
Use of the REVERT(®) total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states.
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DOI：
10.1016/j.mce.2018.01.015
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发表时间：
2018-09-15
影响因子：
4.1
通讯作者：
Gibbs RB
中科院分区：
文献类型：
--
作者：
Kirshner ZZ;Gibbs RB
蛋白质印迹通常用于量化组织中靶蛋白水平的差异。标准方法通常使用管家蛋白的测量来控制加载和蛋白质转移的变化。然而，当管家蛋白也受到实验条件（例如损伤、疾病和/或性腺激素操作）的影响时，这是有问题的。我们的目标是评估一种替代的、也许是更好的方法，对具有不同生理相关性性激素状态的大鼠的脑组织匀浆进行蛋白质印迹分析。从年轻成年雌性大鼠的海马体、额叶皮质和纹状体中收集组织，这些雌性大鼠要么被切除卵巢以模拟手术绝经，要么用卵毒素 4-乙烯基环己烯二环氧化物 (VCD) 处理以模拟过渡性绝经。还从具有正常动情周期的大鼠中收集组织，这些大鼠在发情前期（雌二醇水平高）和动情间期（雌二醇水平低）处死。对 α-微管蛋白、β-肌动蛋白和 GAPDH 进行蛋白质印迹检测，并与荧光总蛋白染色剂 (REVERT®) 比较灵敏度和可靠性。结果表明，与 α-微管蛋白、β-肌动蛋白或 GAPDH 相比，样品间总蛋白染色的变化要小得多，并且具有更大的线性范围。该染色剂稳定且易于使用，并且不干扰管家蛋白的免疫检测或多重检测。此外，我们发现，将数据归一化为总蛋白，而不是归一化为 GAPDH，揭示了海马中 α-微管蛋白表达作为治疗函数的显着差异，并且当数据归一化为总蛋白时，在测量在多个凝胶上运行的配对样品之间的差异时，凝胶到凝胶的一致性明显优于归一化为 GAPDH 时。这些结果表明，REVERT® 总蛋白染色剂可用于脑组织匀浆的蛋白质印迹分析，以控制上样和蛋白质转移的变化，并且与使用管家蛋白来量化多种目标蛋白水平的变化相比，具有显着的优势。
Western blot is routinely used to quantify differences in the levels of target proteins in tissues. Standard methods typically use measurements of housekeeping proteins to control for variations in loading and protein transfer. This is problematic, however, when housekeeping proteins also are affected by experimental conditions such as injury, disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative and perhaps superior method for conducting Western blot analysis of brain tissue homogenates from rats with distinct physiologically relevant gonadal hormone states. Tissues were collected from the hippocampus, frontal cortex, and striatum of young adult female rats that either were ovariectomized to model surgical menopause, or were treated with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause. Tissues also were collected from rats with a normal estrous cycle killed at proestrus when estradiol levels are high, and at diestrus when estradiol levels are low. Western blot detection of α-tubulin, β-actin, and GAPDH was performed and were compared for sensitivity and reliability with a fluorescent total protein stain (REVERT®). Results show that the total protein stain was much less variable across samples and had a greater linear range than α-tubulin, β-actin, or GAPDH. The stain was stable and easy to use, and did not interfere with the immunodetection or multiplexed detection of the housekeeping proteins. In addition, we show that normalization of our data to total protein, but not to GAPDH, revealed significant differences in α-tubulin expression in the hippocampus as a function of treatment, and that gel-to-gel consistency in measuring differences between paired samples run on multiple gels was significantly better when data were normalized to total protein than when normalized to GAPDH. These results demonstrate that the REVERT® total protein stain can be used in Western blot analysis of brain tissue homogenates to control for variations in loading and protein transfer, and provides significant advantages over the use of housekeeping proteins for quantifying changes in the levels of multiple target proteins.
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影响因子：
2.9
作者：
Gilda JE;Gomes AV
通讯作者：
Gomes AV
影响因子：
4
作者：
Flamini, M. I.;Sanchez, A. M.;Simoncini, T.
通讯作者：
Simoncini, T.
DOI：
10.1073/pnas.74.12.5350
发表时间：
1977-01-01
影响因子：
11.1
作者：
ALWINE, JC;KEMP, DJ;STARK, GR
通讯作者：
STARK, GR
影响因子：
1
作者：
KONISHI, S
通讯作者：
KONISHI, S
影响因子：
4.1
作者：
Kumar, Anita;Dumasia, Kushaan;Balasinor, N. H.
通讯作者：
Balasinor, N. H.