Patterson Abigail R, Crawford Alexandra P, Hatcher Amanda S, Ponnoth Dovenia S, Asano Shinichi
Department of Chemistry and Biochemistry, West Virginia Wesleyan College, Buckhannon, WV, USA.
Department of Biomedical Sciences, West Virginia School of Osteopathic Medicine, Lewisburg, WV, USA.
Biochem Biophys Rep. 2025 Apr 14;42:102018. doi: 10.1016/j.bbrep.2025.102018. eCollection 2025 Jun.
Housekeeping protein expression in tissue samples is often used to normalize immunoblotting data. However, utilizing common loading control proteins in pathological conditions without validation can be problematic. Here, we describe the alteration of commonly used loading control protein expressions in asthmatic lung samples and present a simpler and more reliable loading control for western blot analysis. Lung samples from control and asthmatic mice groups were used to assess the expression levels of three commonly used housekeeping proteins (beta-actin, alpha-tubulin, and glyceraldehyde-3-phosphate dehydrogenase). Ponceau S staining data was also obtained to assess the consistency of protein loading. Housekeeping protein expressions varied dramatically in asthmatic groups, ranging from 26 to 278 % compared to the control group ( < 0.05). On the other hand, no significant differences in Ponceau S staining were observed among the groups. There were no differences in protein extraction from lung samples among the groups, but significant differences were observed in bronchoalveolar lavage leukocyte populations ( < 0.05). Our data suggest that lung resident immune cells were dramatically altered in asthma groups, and protein expression of commonly used loading control proteins are unreliable for western blot analysis of asthmatic lungs. We recommend Ponceau S staining as a more reliable loading control for asthmatic lung samples to normalize western blot data.
组织样本中的管家蛋白表达常被用于标准化免疫印迹数据。然而，在未经验证的病理条件下使用常见的上样对照蛋白可能会出现问题。在此，我们描述了哮喘肺样本中常用上样对照蛋白表达的变化，并提出了一种更简单、更可靠的用于蛋白质印迹分析的上样对照。来自对照组和哮喘小鼠组的肺样本用于评估三种常用管家蛋白（β-肌动蛋白、α-微管蛋白和甘油醛-3-磷酸脱氢酶）的表达水平。还获得了丽春红S染色数据以评估蛋白上样的一致性。哮喘组中管家蛋白的表达变化显著，与对照组相比，变化范围为26%至278%（P<0.05）。另一方面，各组之间丽春红S染色未观察到显著差异。各组之间从肺样本中提取蛋白质没有差异，但在支气管肺泡灌洗白细胞群体中观察到显著差异（P<0.05）。我们的数据表明，哮喘组中肺驻留免疫细胞发生了显著变化，常用上样对照蛋白的蛋白表达对于哮喘肺的蛋白质印迹分析是不可靠的。我们建议将丽春红S染色作为哮喘肺样本更可靠的上样对照，以标准化蛋白质印迹数据。
