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

4 C. Elution flow rate was 0.751.0 ml/min and fractions of 5 ml were collected. Elution of βLG variants was monitored at 280 nm with a UV detector (Bio-Rad BioLogic LP workstation, Model 2128).

Protein concentrations of the eluted fractions were estimated by measuring the optical absorption of the protein fractions at 280 nm (Shimadzu UV-1201). The eluted fractions were analyzed by analytical SDS-PAGE and isoelectric focusing (IEF). According to these results, two combined fractions were each pooled and dialyzed against deionized water overnight at 4 C. After dialysis, the two fractions were concentrated by lyophilization (Christ LMC-1, model beta 216), and then analyzed further by using analytical SDS-PAGE, isoelectric focusing (IEF), and immunoblotting (IB).

ANALYTICAL ELECTROPHORESIS
The molecular weights of the proteins in the two fractions, separated with the Model 491 Prep Cell, were estimated by SDS-PAGE under reducing conditions modifying the method of Laemmli.31 Briefly, gels containing a 1220% acrylamide separating gel and a 3% stacking gel were loaded with the samples (0.55 g protein/well), mixed with sample buffer (1:1), and 10 l sample was injected per well. The gels were run (PROTEAN II electrophoresis cell) at 20 mA constant current, 3 W, 185 Vh at RT with 0.024 M Tris/0.192 M glycine/0.1% SDS, pH 8.3 as a running buffer. Bovine βLG containing the A and B variants as also the A and the B variant (Sigma) and molecular weight standards (Bio-Rad SDS-PAGE low range and kaleidoscope polypeptide standards) were run simultaneously. The gels were stained with 0.025% Coomassie Brilliant Blue R-250 (B-0149, Sigma Chemical Co., St. Louis, MO, USA) in 10% isop
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