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J.S. Crabb, K. Renganathan, X. Gu, K.A. West, S.K. Bhattacharya, Z. Wu, J.W. Crabb; Fractionation of Retina for Proteomic Analysis. . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5154.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Detection of integral membrane proteins by 2D–PAGE can be problematic because proteins like rhodopsin usually remain embedded in the first dimension immobilized pH gradient. Here we explore the efficacy of solution state isoelectricfocusing (IEF) and one–dimensional SDS polyacrylamide gel electrophoresis (1D PAGE) for fractionation of bovine retina for proteomic analysis. Methods:Bovine retinal protein extraction efficiency was evaluated in 7M urea and 2M thiourea containing one of eight different detergent solutions. Rhodopsin extraction efficiency was determined by Western analysis. Retinal protein recovery following solution state IEF in 3% ASB–14, 4% CHAPS, or 3% dodecyl maltoside was quantified by the Bradford and bicinchoninic assays. Following solution state IEF using the Multicompartment Electrolyzer (MCE, Proteome Systems) and 1D PAGE, bands were excised, digested in situ with trypsin and proteins identified by capillary LC MS/MS. Results:Retinal extracts in 3% ASB–14 yielded less high mass aggregates of rhodopsin and greater protein recovery in IEF. Solution state IEF in 3% ASB–14 separated retinal proteins into pI fractions 3–5, 5–6.5, 6.5–8, and 8–11. After IEF, 1D–PAGE revealed different electrophoretic profiles for each trapping chamber fraction. A total of 352 proteins were identified from mass spectrometric analyses of select 1D gel bands, including rhodopsin, 31 other integral membrane proteins and 33 membrane–associated proteins. Conclusions: For proteomic studies of retina, solution state IEF followed by 1D–PAGE provides an attractive alternative to 2D gel analyses with immobilized pH gradients. In this study, ASB–14 was the most effective detergent for the solubilization and extraction of retina and identification of rhodopsin.
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