Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, FL 32610, United States of America.
Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, FL 32610, United States of America.
J Pharmacol Toxicol Methods. 2021 Nov-Dec;112:107117. doi: 10.1016/j.vascn.2021.107117. Epub 2021 Aug 30.
The western blot (WB) is the predominate method for protein quantification, frequently used in pharmacological and toxicological studies. To control for technical variation, WB signals are normalized through immunodetection of an internal standard "house-keeping" gene or total protein quantification via staining of the same blot or a duplicate, sister blot. Increasing evidence suggests that house-keeping genes are subject to change after drug treatment or under disease states, causing protein quantification errors in WB. Recent advances in automated capillary-based WB technologies enable measurement of the protein of interest, internal standards, and total protein in a single capillary. Using this approach, we quantified cytochrome P450 3A4 (CYP3A4) across 179 liver samples and compared normalization by both β-actin and total protein to determine which better functions as an internal standard. CYP3A4 is responsible for metabolism of a wide array of xenobiotics and is known to exhibit large inter-person variation, making it a good candidate to evaluate protein quantification. We observed significant differences in β-actin protein levels between liver samples (~20-fold) and found better correlation between CYP3A4 protein and mRNA using total protein normalization than β-actin, indicating total protein normalization to be less error prone for estimation of CYP3A4. Furthermore, by using total protein normalization, we confirmed significant association between CYP3A4 protein expression and the functional CYP3A4 variant CYP3A4*22, which contains two linked SNPs rs35599367 and rs62471956. Our results indicate that the automatic capillary WB instrument combined with total protein normalization provides a high throughput and robust approach for protein quantification.
蛋白质印迹法（WB）是蛋白质定量的主要方法，常用于药理学和毒理学研究。为了控制技术变化，通过免疫检测内参“管家”基因或通过同一张印迹或重复印迹的总蛋白定量，WB 信号被标准化。越来越多的证据表明，管家基因在药物治疗或疾病状态下会发生变化，导致 WB 中的蛋白质定量错误。最近自动化毛细管 WB 技术的进展使得可以在单个毛细管中测量感兴趣的蛋白质、内参和总蛋白。使用这种方法，我们对 179 个肝样本中的细胞色素 P450 3A4（CYP3A4）进行了定量，并比较了β-肌动蛋白和总蛋白的归一化，以确定哪种更好地作为内参。CYP3A4 负责代谢广泛的外源化学物质，并且已知个体间差异很大，使其成为评估蛋白质定量的良好候选物。我们观察到肝样本中β-肌动蛋白蛋白水平存在显著差异（约 20 倍），并且使用总蛋白归一化比β-肌动蛋白更好地显示 CYP3A4 蛋白与 mRNA 之间的相关性，表明总蛋白归一化对于估计 CYP3A4 错误较少。此外，通过使用总蛋白归一化，我们确认了 CYP3A4 蛋白表达与功能性 CYP3A4 变体 CYP3A4*22 之间的显著关联，该变体包含两个连锁 SNP rs35599367 和 rs62471956。我们的结果表明，自动毛细管 WB 仪器结合总蛋白归一化提供了一种高通量且稳健的蛋白质定量方法。
