Gilda Jennifer E, Gomes Aldrin V
Department of Neurobiology, Physiology, and Behavior, University of California, Davis, 191 Briggs Hall, One Shields Avenue, Davis, CA, 95616, USA.
Methods Mol Biol. 2015;1295:381-91. doi: 10.1007/978-1-4939-2550-6_27.
Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on both gels and membranes. Here, we describe the theory and use of stain-free gels for total protein quantification and normalization of Western blots.
蛋白质印迹法是一种常用的实验室技术，用于对蛋白质含量进行半定量。在定量蛋白质表达时，使用上样对照来考虑上样到凝胶上的总蛋白量差异非常重要。常见的上样对照包括通过蛋白质印迹法定量的管家蛋白，如β-肌动蛋白或甘油醛-3-磷酸脱氢酶，或使用考马斯亮蓝或丽春红S等染色剂定量的总蛋白。一种最近开发的总蛋白定量方法采用免染技术，该技术具有线性动态检测范围，可在凝胶和膜上进行蛋白质检测。在这里，我们描述了免染凝胶用于总蛋白定量和蛋白质印迹法标准化的原理及应用。
