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Quantification of MFGM proteins in buttermilk and butter serum by means of a stain free SDS-PAGE method

Holzmüller, Wolfgang, Kulozik, Ulrich
Subtropical plant science 2016 v.49 pp. 102-109
blood serum, butter, buttermilk, cream, fat globules, fluorescence, glycosylation, membrane proteins, milk fat, molecular weight, polyacrylamide gel electrophoresis, skim milk, staining, tryptophan, ultraviolet radiation, whey
In comparison to skim milk and whey, buttermilk and butter serum contain high amounts of functional milk fat globule membrane (MFGM) proteins. Quantification of MFGM proteins is difficult because no analytical standard material is available. By means of conventional SDS-PAGE with subsequent staining, MFGM proteins are analytically separated. However, due to various protein structures (e.g., glycosylation) the affinity to the dye is inconsistent. A direct correlation between measured band intensity and protein concentration and, therefore, absolute protein quantification is not possible. The purpose of this study was to apply a novel stain free SDS-PAGE technique that relies on measuring UV light fluorescence of tryptophan in order to quantify MFGM proteins. Thus, proteins can be quantified without the need for analytical standards, provided that the relative tryptophan content relative to the molecular weight is known. In this study, buttermilk and butter serum obtained from raw cream were examined and the three most abundant membrane proteins were absolutely quantified. Higher amounts of peripheral MFGM proteins were determined in buttermilk (0.9g/L) and butter serum (2.7g/L), in comparison to integral MFGM proteins (0.3–0.5g/L). Conclusions were drawn regarding the absolute protein amount of MFGM material surrounding native milk fat globules.