Application of capillary electrophoresis in the identification of phenotypes containing the beta-lactoglobulin C variant.

A method for the separation of the beta-LG variants A, B, and C was developed using free zone capillary electrophoresis. A separation buffer consisting of 50 mM 2-(N-morpholino)ethane sulfonic acid (pH 8.0) and polyoxyethylene (20)-sorbitan monolaurate was used for the separation. Milk samples from 424 Jersey cows were phenotyped using this method, and the gene frequencies for beta-LG A, B, and C were .41, .53, and .06, respectively. These frequencies were different from those found in studies of Jersey populations in other countries, where the beta-LG B allele had a significantly higher frequency, and the beta-LG A allele a significantly lower frequency, than those of this present study. The frequency of the beta-LG C allele was intermediate to those of other studies. The concentration of beta-LG in the milk was different among the beta-LG phenotypes and was, from greatest to least: AA, AB, AC, BB, and BC.

Separation of beta-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis.

beta-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the beta-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pKa range (pH 5.3-7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified beta-lactoglobulin A and B proteins and a beta-lactoglobulin AC whey. The method was then used to phenotype beta-lactoglobulin in a sample population of New Zealand Jersey cows.

Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins.

The separation of the four major whey proteins by sodium dodecyl sulphate (SDS)-capillary gel electrophoresis (CGE) is described. Whilst commercially purified whey proteins could be analysed using the recommended protocol, the more complex nature of an acid whey and a reconstituted whey protein concentrate (WPC) powder necessitated considerable refinement of the CGE sample buffer. Individual whey proteins in the acid whey and WPC samples were then also separated and quantitated using capillary zone electrophoresis, polyacrylamide gel electrophoresis (PAGE) and HPLC methods and the results were compared. The values obtained for alpha-lactalbumin (alpha-Lac) and beta-lactoglobulin (beta-Lg) were consistent throughout the various methods, although size-exclusion HPLC, SDS-PAGE and SDS-CGE could not separate the two beta-Lg variants or the glycosylated form of alpha-Lac from the beta-Lg. There was considerable variation in the values for the bovine serum albumin and immunoglobulin determined by the different methods and it was concluded that none of the methods could satisfactorily quantitate all four whey proteins.