College of Basic Medical Sciences, Dalian Medical University, Dalian, 116044, Liaoning, China.
Clinical Laboratory, Dalian Municipal Central Hospital, 826-Xinan Road, Shahekou District, Dalian city, Liaoning, 116033, China.
Sci Rep. 2019 Apr 30;9(1):6649. doi: 10.1038/s41598-019-43039-3.
Western blotting is the most extensively used technique for the identification and characterisation of proteins and their expression levels. One of the major issues with this technique is the loss of proteins from the blotted membrane during the incubation and washing steps, which affects its sensitivity and reproducibility. Here, we have optimised the fixation conditions for immunoblotting and lectin blotting on electroblotted polyvinylidene difluoride and nitrocellulose membranes, using a combination of organic solvents and heating. Loss of proteins from polyvinylidene difluoride membranes was greatly reduced using this approach, the intensity of lectin blotting and immunoblotting was shown to increase 2.8- to 15-fold and 1.8- to 16-fold, respectively, compared with that samples without treated. Using the optimised method, cystic fibrosis transmembrane regulator and hypoxia-inducible factor 1, two difficult-to-analyse proteins with important physiological and pathological roles, were effectively detected. Additionally, it may help the identification of novel diagnostic markers for prostate cancer.
免疫印迹是鉴定和分析蛋白质及其表达水平最常用的技术之一。该技术的主要问题之一是在孵育和洗涤步骤中印迹膜上的蛋白质丢失，这会影响其灵敏度和重现性。在这里，我们通过使用有机溶剂和加热的组合优化了免疫印迹和凝集素印迹在电转移聚偏二氟乙烯和硝酸纤维素膜上的固定条件。使用这种方法大大减少了聚偏二氟乙烯膜上蛋白质的丢失，与未经处理的样品相比，凝集素印迹和免疫印迹的强度分别增加了 2.8 至 15 倍和 1.8 至 16 倍。使用优化的方法，可以有效地检测到囊性纤维化跨膜转导调节因子和缺氧诱导因子 1 这两种具有重要生理和病理作用的难以分析的蛋白质。此外，它可能有助于鉴定前列腺癌的新诊断标志物。
