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S. Schaal, L. Geng, E. Bodek, S. Martin, T. Tezel; Optimization of Protein Extraction for Proteomic Analysis of Bruch’s Membrane . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1396.
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To explore the effectiveness of ten protein extraction and solubilization protocols for the solubilization and fractionation of human Bruch’s membrane for proteomic analysis.
Explants of human Bruch’s membrane were prepared from 6 cadaver eyes using an excimer laser. 6.5 mm circular buttons were further processed for protein extraction using ten different extraction protocols. The protocols differed in terms of detergents used (4% CHAPS, 3% ASB–14, 1% SDS), use of collagen crosslink breaking agent (0.1M, N–phenyl–thiazolium bromide, PTB), and/or use of proteolytic cleavage with 1% pepsin. Solubilized proteins were precipitated with trichloroacetic acid and their amount was determined using bicinchoninic acid assay. Extracted protein amount was normalized to the total protein mass for comparing the efficacy of different methods. Following solution state IEF using a multi–compartment fractionator and 1D PAGE or a 2D–PAGE analysis was performed.
Human Bruch’s membrane explants treated with 0.1M PTB yielded two–fold higher solubilized protein than explants treated only with detergents (45% versus 21% of the total protein mass). Further proteolysis by pepsin yielded a higher protein solubilization (an average increment of 50% in protein extraction after pepsin proteolysis). A combined protocol solubilized approximately 84% of total Bruch’s membrane proteins. Higher protein solubilization decreased the size of the Bruch’s membrane explant required for proteomic analysis down to 6.5 mm.
For proteomic analysis of the Bruch’s membrane, solubilization of proteins prior to IEF is crucial. In this study, the most effective protocol for the solubilization and extraction of proteins involved the usage of PTB and pepsin. Increased protein extraction will result in an increased resolution to study the proteomic differences of Bruch’s membrane.
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