Two-dimensional (2-D) gel electrophoresis of aqueous humour proteins

Purpose. To use the technique of 2-D gel electrophoresis to determine a composite protein profile map of 'normal' aqueous humour, and to compare with that seen in patients with intraocular inflammation. Methods. Aqueous humour was obtained at cataract surgery from patients with age-related cataract and patients with uveitis. For the first dimension IEF, aqueous was mixed with sample buffer and applied to a rehydrated Immobiline DryStrip (pH 4-7, pH 3-10). For the second dimension SDS-PAGE, a precast ExcelGel SDS gel (gradient 8-18) was used. The gels were silver stained and analysed using 2-D image analysis software. Results. A composite 2-D gel was constructed from 20 'normal' aqueous samples and contained approximately 200-250 spots. A number of these spots could be identified by comparing them to known 2-D protein databases (SWISS-2DPAGE, SWISS-PROT, REF52 2D) via the world wide web. Aqueous from patients with Fuchs' heterochromic cyclitis showed an increase in immunoglobulin light chains. Conclusions. 2-D gel electrophoresis is a highly sensitive technique for analysing aqueous humour proteins. The creation of a database of 'normal' aqueous proteins will act as a reference standard for future studies and allow comparison with aqueous proteins from a variety of pathological states.