( method of Bradford 29 at 595 ...)Purification and Characterization of beta-Lactoglobulin Genetic Variants A and B Using Preparative Electrophoresis and Isoelectric Focusing

STANDARDS
βLG variants A, B and A/B from bovine milk (Sigma Chemical Co., St. Louis, MO, USA), bovine a-lactalbumin (Sigma Chemical Co., St. Louis, MO, USA), a mixture of caseins (Sigma Chemical Co., St. Louis, MO, USA), and bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA), 10 mg each were dissolved in 10 ml deionized water. Protein concentrations of the standards were estimated, and the EF samples were prepared as described above. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) low molecular weight standards, kaleidoscope polypeptide standards, and isoelectric focusing (IEF) standards were from Bio-Rad (Bio-Rad, Richmond, CA, USA).

PREPARATIVE CONTINUOUS ELUTION PAGE ELECTROPHORESIS
Buffers used in preparative native PAGE were those described by Ornstein-Davis.³⁰ The electrophoresis equipment used was Bio-Rads Model 491 Prep Cell. The separating gel (110 x 37 mm) was 15% acrylamide/0.4% bisacrylamide in 0.375 M Tris-HCl (final concentration), pH 8.8 (catalyst concentrations in resolving gel were 0.025% APS/0.025% TEMED). The stacking gel (10 x 37 mm) was 3% acrylamide/0.08% bisacrylamide in 0.125 M Tris-HCl (final concentration), pH 6.8. The running buffer was 0.025 M Tris-base, 0.19 M glycine, pH 8.3. Before sampling loading, an electrophoresis field was applied at constant power at 5 W for 30 minutes Thereafter 400 l sample was injected and PE was run at constant power of 12 W for 4,000 Vh for 19 h at
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