Mathews Suresh T, Plaisance Eric P, Kim Teayoun
Department of Nutrition and Food Sciences, 101 Poultry Sci Bldg, 260 Lem Morrison Dr., Auburn, AL, 36849, USA.
Methods Mol Biol. 2009;536:499-513. doi: 10.1007/978-1-59745-542-8_51.
Western blot detection methods have traditionally used X-ray films to capture chemiluminescence. The increasing costs for film, reagents, and maintenance have driven researchers away from darkrooms to more sensitive and technologically advanced digital imaging systems. Cooled charge coupled devices (CCD) cameras capture both chemiluminescence and fluorescence images, with limitations for each detection method. Chemiluminescence detection is highly sensitive and relies on an enzymatic reaction that produces light, which can be detected by a CCD camera that records photons and displays an image based on the amount of light generated. However, the enzymatic reaction is dynamic and changes over time making it necessary to optimize reaction times and imaging. Fluorescent detection with a CCD camera offers a solution to this problem since the signal generated by the proteins on the membrane is measured in a static state. Despite this advantage, many researchers continue to use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum by imaging fluorphores in the infrared spectrum. Infrared imaging is equally sensitive to chemiluminescence and more sensitive to visible fluorescence due in part to reduced autofluorescence in the longer infrared wavelength. Furthermore, infrared detection is static, which allows a wider linear detection range than chemiluminescence without a loss of signal. A distinct advantage of infrared imaging is the ability to simultaneously detect proteins on the same blot, which minimizes the need for stripping and reprobing leading to an increase in detection efficiency. Here, we describe the methodology for chemiluminescent (UVP BioChemi) and infrared (LI-COR Odyssey) imaging, and briefly discuss their advantages and disadvantages.
传统上，蛋白质印迹检测方法使用X射线胶片来捕捉化学发光信号。胶片、试剂和维护成本的不断增加，促使研究人员逐渐摒弃暗室，转而采用更灵敏、技术更先进的数字成像系统。冷却电荷耦合器件（CCD）相机可捕捉化学发光和荧光图像，但每种检测方法都存在一定局限性。化学发光检测灵敏度高，依赖于产生光的酶促反应，该反应产生的光可被CCD相机检测到，相机记录光子并根据产生的光量显示图像。然而，酶促反应是动态的，会随时间变化，因此有必要优化反应时间和成像过程。使用CCD相机进行荧光检测为解决这一问题提供了一种方案，因为膜上蛋白质产生的信号是在静态下测量的。尽管有此优势，但由于荧光团在可见光谱中的性能普遍较差，许多研究人员仍继续使用化学发光检测方法。红外成像系统为化学发光的动态反应以及在可见光谱中检测到的荧光团性能不佳的问题提供了解决方案，它通过对红外光谱中的荧光团进行成像来解决这些问题。红外成像对化学发光同样敏感，对可见荧光更敏感，部分原因是在较长的红外波长下自发荧光减少。此外，红外检测是静态的，与化学发光相比，它允许更宽的线性检测范围且不会损失信号。红外成像的一个显著优势是能够同时检测同一张印迹上的蛋白质，这最大限度地减少了剥离和重新探测的需求，从而提高了检测效率。在此，我们描述了化学发光（UVP BioChemi）和红外（LI-COR Odyssey）成像的方法，并简要讨论了它们的优缺点。
