SEPARATION OF BOVINE BLG PHENOTYPES BY CONTINUOUS ELUTION ELECTROPHORESIS
Figure 1 shows an elution profile of bovine LG A/B separated by preparative continuous electrophoresis as described in Methods. The fractions were analyzed by analytical SDS-PAGE, and then pooled: peak I (fractions 3543) and peak II (fractions 4755) and concentrated. Total protein recovery of the combined fractions was 78% (15.6 mg/20 mg) while protein concentration of peak I (fractions 3543) was 0.190 mg/ml (total 7.05 mg) and that of peak II (fractions 4755) was 0.160 mg/ml (total 8.5 mg).
CHARACTERIZATION OF THE TWO COMBINED FRACTIONS SEPARATED BY THE MODEL
491 PREP CELL
Molecular weights of the pooled fractions analyzed by SDS-PAGE under reducing conditions are shown in Figure 2A. The two eluted fractions each have the same molecular weight as bovine βLG A/B, according to their mobility in SDS-PAGE. The two fractions show only one single band by electrophoresis.
Identification of the combined fractions was confirmed by western blot with polyclonal antibodies to bovine βLG. Immunoblotting shows that βLG A/B and both eluted fractions are recognized by antiserum to βLG (Figure 2B).
Since IEF is the most sensitive method for charge distinction within
different phenotypes, the isoelectric points of the two eluted fractions,
separated by continuous native PAGE, were estimated also by isoelectric
focusing at pH range 58. Commercial bovine βLG phenotype standards A,
B, and A/B were analyzed