Martin Karen, Hart Courtenay, Schulenberg Birte, Jones Laurie, Patton Wayne F
Proteomics Section, Molecular Probes, Inc., Eugene, Oregon 97402, USA.
Proteomics. 2002 May;2(5):499-512. doi: 10.1002/1615-9861(200205)2:5<499::AID-PROT499>3.0.CO;2-H.
A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.
本文描述了一种双色荧光检测方法，该方法基于将BODIPY TR-X染料的琥珀酰亚胺酯共价偶联到固定在聚偏二氟乙烯膜上的蛋白质上，然后使用荧光性沉淀底物ELF 39-磷酸与碱性磷酸酶偶联的报告分子相结合来检测目标蛋白质。这使得图谱中的所有蛋白质都呈现为红色荧光信号，而用碱性磷酸酶偶联物特异性检测到的蛋白质则呈现为绿色荧光信号。这种双色检测系统与配备照相或电荷耦合器件相机的紫外落射或透射照明器以及配备适当激发/发射滤光片的氙弧光源广泛兼容。双色法能够检测低至纳克量的蛋白质，并允许在单个电印迹上相对于整个蛋白质图谱明确鉴定目标蛋白质，从而无需运行重复的凝胶，否则这些凝胶需要通过银染来可视化总蛋白质，并随后与电印迹上产生的化学发光或比色信号进行比对。将该检测方法与Alexa Fluor 350染料偶联的单克隆抗体相结合，可以在同一电印迹上同时对两种抗原和总蛋白质图谱进行荧光检测。
