Richards D P, Stathakis C, Polakowski R, Ahmadzadeh H, Dovichi N J
Department of Chemistry, University of Alberta, Edmonton, Canada.
J Chromatogr A. 1999 Aug 20;853(1-2):21-5. doi: 10.1016/s0021-9673(99)00687-1.
We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFP's FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.
我们使用毛细管等电聚焦结合激光诱导荧光检测（cIEF-LIF）研究了荧光标记对蛋白质等电点（pI值）的影响。具体而言，我们用荧光染料3-(2-呋喃甲酰基)喹啉-2-甲醛（FQ）标记了来自维多利亚多管水母的绿色荧光蛋白（GFP）。cIEF-LIF用于监测GFP的天然荧光，并显示了GFP的FQ标记产物的pI变化。用FQ对GFP进行多次标记产生了一系列pI值向低pH偏移的产物。我们用传统的平板凝胶IEF验证了cIEF-LIF的结果。我们的cIEF-LIF技术能够常规检测到10⁻¹¹ M的FQ标记蛋白，而采用银染检测的传统平板凝胶IEF在相同样品中的检测限为10⁻⁷ M。
