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C. Garcia, N. Patel, S. K. Bhattacharya; Strategies to Recover Proteins From Ocular Tissues for Proteomics. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3795.
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© ARVO (1962-2015); The Authors (2016-present)
To determine optimal protein extraction strategies from different ocular tissues. To determine optimal detergents for extraction of proteins from the different regions of the eye and whether repertoire of proteins varies with extraction strategies.
Proteins were extracted from tissues (for example, cornea, lens, anterior and posterior sclera and retina) derived from enucleated pig eyes (from Tucker Packing company, Orville, Ohio or from department of surgery, UM Miller School of Medicine) in 125 mM Tris-Cl buffer pH 7.0, 100 mM NaCl and 35 different detergents (ionic, nonionic and zwitterionic categories). The protein concentrations were determined using the Bradford Assay and SDS-PAGE were performed using 4-20% gradient mini gels. In other experiments, parameters such as NaCl concentration and temperature were varied. These studies were performed adhering to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Freshly enucleated pig eyes (n=8 for each detergent for 35 detergents) showed differences in their extraction capacity for a given region of the eye ranging from 2.3 to 50.7 µg protein/mg of ocular tissue. Lens allowed maximum soluble protein extraction (50.7 µg) per mg of tissue. ASB-14 was found to be the best detergent for anterior and posterior sclera (n=10 eyes). Anterior and posterior sclera presented different repertoire of proteins as revealed by the Coommassie blue stained SDS-PAGE protein profile. Relatively higher protein yield was found from posterior sclera (10.8 µg/mg of tissue) than anterior sclera (8.9 µg/mg of tissue) using ASB-14.
Different regions of the eye are best extracted in different detergents. For example, three best detergents in terms of protein yield for cornea are ASB-14, SDS and n-Nonyl-ß-D-glucopyranoside while that for sclera are ASB-14, n-Octyl-ß-D-glucopyranoside and CHAPS. The extraction strategy may affect the final outcome in protein profiling by mass spectrometry or by other methods.
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