SAMPLE PREPARATION AFFECTS SEPARATION OF WHEY PROTEINS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Measured concentrations of whey proteins in single milk samples often differ by more than 20% when analyzed by different separation methods. In the current study, we examined the effects of using guanidine hydr ochloride or urea at zero and 8.5 h after sample preparation and the e ffects of using dithiothreitol or a-mercaptoethanol on the separation of bovine milk proteins by reversed-phase high-performance liquid chro matography. Treatment with guanidine hydrochloride or urea resulted in similar separation of milk proteins when samples were injected immedi ately after preparation. Separation was repeatable over 8.5 h for samp les prepared with guanidine hydrochloride, whereas the resolution of a -lactalbumin and beta-lactoglobulin decreased for samples treated with urea. Treatment with dithiothreitol improved the resolution of alpha- lactalbumin and beta-lactoglobulin in comparison with treatment with 2 -mercaptoethanol. Quantitation of whey proteins is more reliable when milk samples are treated with dithiothreitol and guanidine hydrochlori de than when treated with 2-mercaptoethanol or urea.