Protein normalization of western blots has relied upon housekeeping proteins which exhibit signal saturation and varied cellular expression level variations. These issues can produce spurious results leading to erroneous conclusions. A superior method to protein normalization using housekeeping proteins is Total Protein Normalization, a method now recognized as the gold standard for quantitative westerns. Total Protein Normalization requires that all proteins on a membrane be stained or labeled uniformly, imaged, and then analyzed for total protein. It is important that such a normalization process not interfere with typical immunodetection methods, fits within existing western workflows, and exhibits a linear relationship of signal intensity to protein load under all experimental conditions. Here we report that we developed a new reagent enabling Total Protein Normalization, and we demonstrate its superior protein normalization capabilities through analysis of target proteins in different cell backgrounds. These data illustrate how housekeeping proteins exhibit signal saturation, yield erroneous normalization data, and display sample-to-sample variations averaging 48.2 % overall. Signal intensities obtained using our new method show a linear relationship to protein sample load, thus providing accurate protein normalization with an overall average variation of 7.7 %.

蛋白质印迹的蛋白质标准化依赖于表现出信号饱和和不同细胞表达水平变化的管家蛋白质。这些问题可能会产生导致错误结论的虚假结果。使用看家蛋白进行蛋白质标准化的一种更好的方法是总蛋白质标准化，这种方法现在被公认为定量西方的黄金标准。总蛋白归一化要求膜上的所有蛋白均被染色或标记，成像，然后分析总蛋白。重要的是，这样的标准化过程不会干扰典型的免疫检测方法，适合现有的西方工作流程，并在所有实验条件下表现出信号强度与蛋白质负载的线性关系。在这里，我们报告说我们开发了一种新试剂，可实现总蛋白质标准化，并通过分析不同细胞背景中的目标蛋白质证明了其卓越的蛋白质标准化能力。这些数据说明了看家蛋白如何表现出信号饱和、产生错误的归一化数据，并显示样本间差异平均为 48.2%。使用我们的新方法获得的信号强度显示出与蛋白质样品负载的线性关系，从而提供准确的蛋白质标准化，总体平均变异为 7.7%。并显示样本与样本之间的差异，总体平均为 48.2%。使用我们的新方法获得的信号强度显示出与蛋白质样品负载的线性关系，从而提供准确的蛋白质标准化，总体平均变异为 7.7%。并显示样本与样本之间的差异，总体平均为 48.2%。使用我们的新方法获得的信号强度显示出与蛋白质样品负载的线性关系，从而提供准确的蛋白质标准化，总体平均变异为 7.7%。