Tarlton J F, Knight P J
Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol, BS18 7DY, United Kingdom.
Anal Biochem. 1996 May 15;237(1):123-8. doi: 10.1006/abio.1996.0209.
The quantitation of stained Western blots by reflectance and transmission scanning was explored using a blot of monoclonal immunoglobulin G probed with anti-mouse antibody. The so-called "scanned absorbance" output from a document scanner was found to be directly proportional to the fraction of light absorbed rather than obeying the logarithmic relationship expected for true spectrophotometric absorbance. This explains observations in the literature of a strongly curved relation between loading and "absorbance." Laser transmission densitometry of a blot immersed in a clarifying solvent mixture showed that peak area was linearly related to loading over a wide range. In reflectance mode the document scanner also gave equally linear quantitation of dry blots, providing that a logarithmic correction curve was applied during scanning. It was found advantageous to interpose a red acetate filter sheet between the blot and the scanning table to aid detection of weakly stained bands. The document scanner gave less satisfactory results when used in transmission mode on a clarified blot because weak bands were poorly quantitated.
使用抗小鼠抗体探测的单克隆免疫球蛋白G印迹，通过反射和透射扫描对蛋白质印迹染色进行定量分析。发现文档扫描仪输出的所谓“扫描吸光度”与吸收光的比例成正比，而不是遵循真正分光光度吸光度预期的对数关系。这解释了文献中关于上样量与“吸光度”之间强曲线关系的观察结果。将印迹浸入澄清溶剂混合物中进行激光透射密度测定表明，在很宽的范围内峰面积与上样量呈线性关系。在反射模式下，文档扫描仪同样能对干印迹进行线性定量分析，前提是在扫描过程中应用对数校正曲线。发现在印迹和扫描台之间插入一张红色醋酸纤维滤片有助于检测弱染色条带。当在透射模式下对澄清的印迹使用文档扫描仪时，结果不太令人满意，因为弱条带的定量效果不佳。
