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PS Sexton, RJ Cenedella; Use of Dynabeads® M500Subcellular to Provet hat Lens Membrane Steroid Binding Proteinisa Microsomal Protein . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4634.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To identify the subcellular fraction of lens epithelium which contains the membrane steroid binding protein (MSBP). MSBP is proposed to mediate rapid non-genomic effects of steroid hormones which may underlie steroid induced cataracts. Methods:A "microsomal" fraction was prepared from fresh rat liver and lens epithelium by homogenization in 0.25 M sucrose and differential centrifugation. Dynabeads®M500 Subcellular were used to isolate the microsomal fraction from these subcellular preparations. Dynabeads coated with an un-conjugated goat anti-rabbit IgG secondary antibody and a primary polyclonal antibody to a MSBP carboxy-terminal peptide were incubated with subcellular fractions from rat liver and lens epithelium and the bound material was separated by SDS-PAGE. MSBP is membrane anchored through its amino-terminus. Extracted material was probed by Western blot with an antibody to HMG CoA Reductase (HMGR), a known microsomal protein, as well as antibodies to MSBP and VLA-2 , the latter specific for plasma membrane. Results:Microsomes from rat liver, and rat lens epithelium reacted strongly with anti-HMGR and the rat liver microsomal preparation reacted with the plasma membrane antibody, VLA-2 . Dynabeads M500 with attached secondary and primary antibodies as described above bound a lens subcellular fraction which reacted with anti-HMGR and MSBP but did not react with the plasma membrane antibody. Dynabeads with attached secondary antibody only did not bind microsomes. Although HMGR has also been found in peroxisomes of liver, this fraction was removed by the differential centrifugation proceedure. Conclusion:This is the first report describing the use of Dynabeads (M500) as an effective and precise method of isolating microsomes from a of mixed subcellular preparation. Membrane steroid binding protein in the rat lens and liver is present in microsomes.
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