Fowler S D, Brown W J, Warfel J, Greenspan P
Department of Pathology, School of Medicine, University of South Carolina, Columbia.
J Lipid Res. 1987 Oct;28(10):1225-32.
We describe the use of the fluorescent dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, as a general-purpose reagent for the rapid detection and quantitation of a wide variety of lipids and other hydrophobic compounds separated by thin-layer chromatography. After samples are applied to silica gel plates and chromatographed, the plate is briefly dipped into a nile red solution (8 micrograms/ml of methanol-water 80:20, v/v). Background fluorescence of nile red dye adsorbed to the silica gel is then preferentially destroyed by dipping the plate in a dilute aqueous solution of bleach. After drying, lipid bands are visualized under ultraviolet light. Reflectance fluorometry (Ex: 580 nm; Em: 640 nm) is utilized for in situ quantitative analysis of the fluorescence of the lipids on the nile red-stained plate. Neutral lipids, phospholipids, sphingolipids, and fatty acids can be examined, although the nile red fluorescence intensity varies significantly among the lipid classes. Also, staining is stronger for unsaturated lipids than for saturated lipids. The lower detection limit of the assay is 25-100 ng for cholesterol, cholesteryl esters, triacylglycerols, and phospholipids.
我们描述了使用荧光染料尼罗红（9-二乙氨基-5H-苯并[α]吩恶嗪-5-酮）作为通用试剂，用于快速检测和定量通过薄层色谱法分离的多种脂质及其他疏水化合物。将样品点样于硅胶板上并展开后，把薄板短暂浸入尼罗红溶液（8微克/毫升，甲醇-水，80:20，体积比）中。然后将薄板浸入稀漂白水溶液中，优先破坏吸附在硅胶上的尼罗红染料的背景荧光。干燥后，脂质条带在紫外光下可见。利用反射荧光法（激发波长：580纳米；发射波长：640纳米）对尼罗红染色薄板上的脂质荧光进行原位定量分析。中性脂质、磷脂、鞘脂和脂肪酸均可检测，尽管尼罗红荧光强度在不同脂质类别间差异显著。此外，不饱和脂质的染色比饱和脂质更强。该检测方法对胆固醇、胆固醇酯、三酰甘油和磷脂的检测下限为25 - 100纳克。
