Gerk Phillip M
Dept. of Pharmaceutics, Virginia Commonwealth University, MCV Campus, School of Pharmacy, Room 454F Smith Bldg, 410 N. 12th Street, P.O. Box 980533, Richmond, VA 23298–0533, USA.
J Pharmacol Toxicol Methods. 2011 May-Jun;63(3):279-82. doi: 10.1016/j.vascn.2011.01.003. Epub 2011 Jan 26.
Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots.
The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-binding cassette transporter isoform C2/multidrug resistance-associated protein 2 (ABCC2/MRP2).
Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotted with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data.
The limits of quantitation for the time-insensitive technique described here were from 0.001 μg to 0.5 μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique were demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance.
The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.
为克服基于胶片的化学发光免疫印迹检测的局限性，需要对参与药物转运和处置的蛋白质表达水平进行定量分析。
描述并验证一种用于检测ATP结合盒转运体C2亚型/多药耐药相关蛋白2（ABCC2/MRP2）的定量免疫荧光印迹法。
通过对从过表达MRP2的Sf9细胞中分离的膜囊泡蛋白进行电泳，随后用红外标记的二抗进行印迹，来进行蛋白质免疫印迹分析。使用奥德赛红外成像系统（Li-Cor；内布拉斯加州林肯市）检测结合的复合物。使用奥德赛应用软件对图像进行分析以获得积分强度，然后对强度数据进行线性回归。
此处所述的时间不敏感技术的定量限为0.001μg至0.5μg总膜蛋白，斜率的变异系数为8.9%；r2值为0.986±0.012。该技术的实用性和灵敏度在定量人胎盘组织样本中MRP2的表达时得到了证明，其中MRP2的丰度较低。
所述的免疫荧光印迹技术可对诸如MRP2等大型整合膜蛋白进行灵敏、可重复的定量测定，且从长远来看可能节省成本。
