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G Surh, J Ghosh, JI Clark; Analyzing the Proteins of the Mouse Lens Using Laser Capture Microdissection (LCM) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3583.
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Purpose: Laser capture microdissection was used for the analysis of the protein constituents of cells at the extremes of the anterior-posterior pole of the lens in wildtype and alpha-B knockout transgenic mice (generously provided by E. Wawrousek, of the NEI). The protein composition of the anterior cortical lens fibers was compared with the corresponding regions of posterior cortical fibers using SDS polyacrylamide gel electrophoresis. Methods: Lenses were removed from wildtype and alpha-B knockout mice and frozen for sectioning. Sections were cut at a thickness of 10-20 microns. Cell nuclei of the anterior epithelium and the bow region of the equator were identified and used as landmarks to determine the anterior-posterior pole of the lens. Once established, samples were collected from anterior cortical and posterior cortical fibers using the PixCell II Laser Capture Microdissection (LCM) System (Arcturus Engineering Inc, Mountain View, CA, USA). Protein profiles were produced and analyzed using the NuPAGE Bis-Tris Gel Electrophoresis System (Invitrogen) and 4-12% gradient gels. Results: LCM was very efficient for the selective procurement of cell samples as required for this study. In all samples there were prominent broad bands at a M.W. of approximately 20 kDa representing crystallins and a smaller band at approximately 108kDa. The proteins in the anterior cortex of the wildtype and alpha-B knockout were similar except that the 108 kDa component was a more prominent constituent in the posterior cortex of the alpha-B knockout than in the posterior cortex of the wild type animals. Conclusions: LCM combined with SDS-PAGE is a promising approach for the study of protein distributions in layers of lens fibers. The preliminary results of this study indicated an asymmetric spatial distribution of proteins along the length of lens fibers in wildtype and alpha-B knockout transgenic mice. In addition, a high molecular weight component of approximately 108 kDa may be abnormally expressed in lenses in which alpha-B expression is disrupted. We acknowledge grant # EY04742 for support.
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