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Purification and Characterization of beta-Lactoglobulin Genetic Variants A and B Using Preparative Electrophoresis and Isoelectric Focusing

ropanol/7% acetic acid, and were destained with 50% isopropanol/10% acetic acid.

ANALYTICAL IEF
The isoelectric points of the two βLG fractions separated on the Model 491 Prep Cell, were analyzed by modifying the method obtained from Bio-Rad (IEF Ready Gel Application Guide). IEF was performed on Bio-Rad IEF Ready Gels (pH 58) with 20 mM NaOH as a cathode buffer and 7 mM phosphoric acid as an anode buffer on Bio-Rads Mini Prep Cell apparatus. The two βLG fractions and the standards were dissolved in distilled water containing 10% glycerol (1 mg/ml). The run was performed at 5 W constant power and 500 Vmax. for 2.5 h. The gels were stained and destained as described above.

IMMUNOBLOTTING
The two βLG fractions, from preparative electrophoresis, were also characterized by polyclonal antiserum to βLG (Nordic Immunologic, Tilburg, Netherlands). Standards were as described above. Sample proteins, separated by PAGE or IEF, were electrophoretically blotted into nitrocellulose (0,45 m nitrocellulose membrane, Hybond-C, Amersham) by the method of Towbin.32 The electrophoretic blotting was performed with 25 mM Tris, 192 mM glycine, pH 8.3, at 30 V overnight. Residual binding sites were blocked with 1.0% bacto-gelatin for 2 h at RT, and the nitrocellulose sheet was then washed three times with PBS Tween 20 (0.05%), 10 minutes each. To detect the βLG specific proteins, the nitrocellulose sheet was incubated with the polyclonal antiserum to βLG (5 l/50 ml PBS/0.05% Tween 20) for 2 h at RT, washed three times as above, and then incubated with horseradish peroxidase-coated second antiserum (10 l/50 ml PBS Tween 20 (0.05%) for
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