Identifying new sperm Western blot loading controls
Identifying new sperm Western blot loading controls
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识别新的精子蛋白质印迹上样对照。
DOI：
10.1111/and.14226
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发表时间：
2021-09-03
期刊：
影响因子：
2.4
通讯作者：
Ma，Fang
中科院分区：
文献类型：
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作者：
Feng，Ying;Wang，Ruohan;Ma，Fang
蛋白表达水平的测定在生物学和医学研究中都起着举足轻重的作用。管家蛋白通常由管家基因编码，被用作装载控制蛋白，使蛋白质表达正常化。显然，适当的参考标准对于充分分析蛋白质表达是必不可少的。然而，我们的研究表明，广泛使用的正常化蛋白，其表达水平在精子样本中差异很大，不适合数据标准化。为了揭示在精子中稳定表达的蛋白质，我们分析了一些已发表的精子转录组数据。选择表达水平相对稳定（co - efficient variation value小于0.35）的7个蛋白，并通过定量实时聚合酶链反应（pcr）、Western Blot （WB）和免疫细胞化学进一步评估。我们的研究结果表明，在经典的管家蛋白中，只有β -微管蛋白在85个个体的精子样本中保持不变。与其他经典的管家蛋白如甘油醛3 -磷酸脱氢酶、肌动蛋白和组蛋白H3相比，Cullin‐1 （CUL1）和F‐box蛋白7 （FBXO7）似乎更适合作为精子蛋白研究中WB的内部对照。结合这些蛋白的位置，CUL1和FBXO7被认为是总蛋白的管家蛋白。
The measurement of protein expression level plays a pivotal role in both biological and medical studies. Housekeeping proteins, generally encoded by housekeeping genes are used as loading control proteins to normalize protein expression. Obviously, proper reference standards are essential for adequate analysis of protein expression. However, our study showed that the widely used normalisation proteins, whose expression levels varied greatly among sperm samples, were unsuitable for data standardisation. To uncover the proteins steadily expressed in sperm, we analysed several published transcriptome data of sperm. Seven proteins whose expression levels were relatively stable (co‐efficient variation values less than 0.35) were selected and further evaluated by quantitative real‐time polymerase chain reaction, Western Blot (WB) and immunocytochemistry. Our results showed that among the classical housekeeping proteins, only β‐tubulin remained constant in sperm samples from 85 individuals. Compared with other classical housekeeping proteins such as glyceraldehyde 3‐phosphate dehydrogenase, actin and histone H3, Cullin‐1 (CUL1) and F‐box only protein 7 (FBXO7) seemed to be more suitable to be used as internal controls for WB in sperm protein studies. Combined with the locations of these proteins, CUL1 and FBXO7 were suggested to be used as a housekeeping protein for total proteins.