Esmaeli-Azad B, Feinstein S C
Department of Biological Sciences, University of California, Santa Barbara 93106.
Anal Biochem. 1991 Dec;199(2):275-8. doi: 10.1016/0003-2697(91)90101-x.
Quantitative interpretation of protein immunoblotting procedures is hampered by a variety of technical liabilities inherent in the use of photographic and densitometric methods. In this paper, we present a novel, simple, and generally applicable alternative procedure to acquire quantitative data from immunoblots. Our strategy employs both the standard alkaline phosphatase color reaction and radiolabelled Protein A. The color reaction is used to localize the polypeptide of interest after transfer to a solid support. The colored bands are then excised and the radioactivity in the colocalized Protein A is quantitated in a gamma counter. In addition to avoiding the problems associated with photographic and densitometric procedures, our assay also overcomes common problems associated with variable gel lane width and individual band distortion. The resulting data is linear over a range of at least 50-fold (10-500 ng of specific protein, for the example used in this study) and is highly reproducible.
蛋白质免疫印迹法的定量解读受到使用照相和光密度测定法所固有的各种技术缺陷的阻碍。在本文中，我们提出了一种新颖、简单且普遍适用的替代方法，用于从免疫印迹中获取定量数据。我们的策略同时采用标准碱性磷酸酶显色反应和放射性标记的蛋白A。显色反应用于在转移到固相支持物后定位目标多肽。然后切下有色条带，并在γ计数器中对共定位的蛋白A中的放射性进行定量。除了避免与照相和光密度测定法相关的问题外，我们的检测方法还克服了与可变凝胶泳道宽度和单个条带变形相关的常见问题。所得数据在至少50倍的范围内呈线性（对于本研究中使用的示例，为10 - 500 ng特定蛋白质），并且具有高度可重复性。
