Isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity.
The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.
Neyestani
Tirang R
TR
Department of Nutrition and Biochemistry, School of Public Health, Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran. tneyestani@umsha.ac.ir
Djalali
Mahmoud
M
Pezeshki
Mohamad
M
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007768:Lactalbumin / Q000276:immunology* / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000276:immunology* / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000276:immunology* / Q000302:isolation & purification*
D007782:Lactoglobulins; D012710:Serum Albumin, Bovine; D007768:Lactalbumin