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X.F. Wang, M.M. Jablonski; Application of Two-dimensional Differential In-gel Electrophoresis (2D-DIGE) to Identify Differentially Expressed Proteins in Cultured Xenopus Retinas . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4559.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In our previous studies of the Xenopus laevis embryonic retina, we have demonstrated that removal of the RPE not only disrupts photoreceptor outer segment assembly, but also differentially regulates the protein expression profiles of both photoreceptors and Müller cells. The traditional candidate protein approach that we have used thus far is inherently labor-intensive and time consuming. To facilitate the delineation of the mechanisms underlying the complex process of outer segment assembly, we have incorporated two-dimensional differential in-gel electrophoresis (2D-DIGE) into our experimental strategies. Using this methodology, three independent pools of proteins can be pre-labeled each with Cydye fluors (e.g. Cy2, Cy3, Cy5) that can be detected at unique wavelengths. The labeled proteins are then mixed and separated on a multiplexed 2D gel. In the present study, the feasibility of 2D-DIGE to detect the differences in protein expression levels between cultured intact Xenopus embryonic retinas and RPE-deprived retinas is presented. Methods: Two pools of retinas were removed from Xenopus embryos at stage 33/34 and the RPE was removed from one pool. Intact and RPE-deprived retinas were cultured in Niu-Twitty medium for 3 days. Proteins were extracted, same amount of total proteins from intact and RPE-deprived retinas were labeled by Cy3 and Cy5, respectively. These two pools of labeled proteins were mixed and separated on the same pI strip and gel for first and second dimension electrophoresis, respectively. DeCyder software was applied to quantify the differences in protein expression profiles between intact and RPE-deprived retinas. Results: Analysis of the 2D image of the protein expression profiles of intact and RPE-deprived retinas identified 335 protein spots in both samples (pI range=4.8-6.8; MW range=14-97 kD). Among the detected protein spots, 16 were up-regulated and 7 were down-regulated by 2-fold in RPE-deprived retinas. Molecular identification of the differentially regulated spots is pending. Conclusion: Because 2D-DIGE technology uses a multiplexing fluorescence approach, it provides more accurate quantification, higher reproducibility, and lower variation of protein expression data than conventional 2D methods. We have demonstrated that 2D-DIGE is a powerful tool for detection of differences in protein expression levels between intact and RPE-deprived retinas. Identication of these differentially expressed proteins may reveal the key molecules involved in regulating photoreceptor outer segment assembly.
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