A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk

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A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk. / Blans, Kristine; Larsen, Lotte Bach; Wiking, Lars; Rasmussen, Jan Trige.

I: Journal of Extracellular Vesicles, Bind 3, Nr. 24214, 30.04.2014, s. 1.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisKonferenceabstrakt i tidsskriftForskning

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@article{a0519831e16a45d886c5bc772796fd13,
title = "A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk",
abstract = "Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre-coloured soluble fraction above the casein pellet were further isolated from casein remnants by size-exclusion chromatography. Isolated membrane vesicles were investigated by electron microscopy, sucrose density centrifugation, western blotting and particle size analysis. Results: A crude phospholipid membrane fraction can be obtained from skim milk by ultracentrifugation. Casein micelle remnants as well as smaller protein components in the crude vesicle fraction can be successfully removed by size chromatography. Electron microscopy of the vesicle isolate reveals circular structures with membrane vesicle character. Sucrose density profiles of the isolate shows an enrichment of the membrane vesicle marker MFG-E8 at density 1.04 g/cm3, but also fractions at higher (up to 1.2 g/cm3) as well as lower density (down to 1.03 g/cm3) exhibits significant MFG-E8 levels. Particle sizes in the range 50–300 nm is observed all over the gradient. The variety of the membrane vesicles is currently being investigated further by several means. Summary/conclusion: A new procedure for easy and gentle isolation of bovine milk membrane vesicles encompassing ultracentrifugation and size-exclusion chromatography has been established. The resulting vesicle isolate exhibits the general membrane vesicle characteristics and provides an appropriate start material from which the variety of milk vesicles can be investigated",
author = "Kristine Blans and Larsen, {Lotte Bach} and Lars Wiking and Rasmussen, {Jan Trige}",
year = "2014",
month = "4",
day = "30",
doi = "10.3402/jev.v3.24214",
language = "English",
volume = "3",
pages = "1",
journal = "Journal of Extracellular Vesicles",
issn = "2001-3078",
publisher = "Co-Action Publishing",
number = "24214",

}

RIS

TY - ABST

T1 - A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk

AU - Blans, Kristine

AU - Larsen, Lotte Bach

AU - Wiking, Lars

AU - Rasmussen, Jan Trige

PY - 2014/4/30

Y1 - 2014/4/30

N2 - Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre-coloured soluble fraction above the casein pellet were further isolated from casein remnants by size-exclusion chromatography. Isolated membrane vesicles were investigated by electron microscopy, sucrose density centrifugation, western blotting and particle size analysis. Results: A crude phospholipid membrane fraction can be obtained from skim milk by ultracentrifugation. Casein micelle remnants as well as smaller protein components in the crude vesicle fraction can be successfully removed by size chromatography. Electron microscopy of the vesicle isolate reveals circular structures with membrane vesicle character. Sucrose density profiles of the isolate shows an enrichment of the membrane vesicle marker MFG-E8 at density 1.04 g/cm3, but also fractions at higher (up to 1.2 g/cm3) as well as lower density (down to 1.03 g/cm3) exhibits significant MFG-E8 levels. Particle sizes in the range 50–300 nm is observed all over the gradient. The variety of the membrane vesicles is currently being investigated further by several means. Summary/conclusion: A new procedure for easy and gentle isolation of bovine milk membrane vesicles encompassing ultracentrifugation and size-exclusion chromatography has been established. The resulting vesicle isolate exhibits the general membrane vesicle characteristics and provides an appropriate start material from which the variety of milk vesicles can be investigated

AB - Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre-coloured soluble fraction above the casein pellet were further isolated from casein remnants by size-exclusion chromatography. Isolated membrane vesicles were investigated by electron microscopy, sucrose density centrifugation, western blotting and particle size analysis. Results: A crude phospholipid membrane fraction can be obtained from skim milk by ultracentrifugation. Casein micelle remnants as well as smaller protein components in the crude vesicle fraction can be successfully removed by size chromatography. Electron microscopy of the vesicle isolate reveals circular structures with membrane vesicle character. Sucrose density profiles of the isolate shows an enrichment of the membrane vesicle marker MFG-E8 at density 1.04 g/cm3, but also fractions at higher (up to 1.2 g/cm3) as well as lower density (down to 1.03 g/cm3) exhibits significant MFG-E8 levels. Particle sizes in the range 50–300 nm is observed all over the gradient. The variety of the membrane vesicles is currently being investigated further by several means. Summary/conclusion: A new procedure for easy and gentle isolation of bovine milk membrane vesicles encompassing ultracentrifugation and size-exclusion chromatography has been established. The resulting vesicle isolate exhibits the general membrane vesicle characteristics and provides an appropriate start material from which the variety of milk vesicles can be investigated

U2 - 10.3402/jev.v3.24214

DO - 10.3402/jev.v3.24214

M3 - Conference abstract in journal

VL - 3

SP - 1

JO - Journal of Extracellular Vesicles

JF - Journal of Extracellular Vesicles

SN - 2001-3078

IS - 24214

ER -