Plasma membrane isolation
AssigneesImec, Katholieke Universiteit Leuven, KU LEUVEN R&D
InventorsDeepak Balaji Thimiri Govinda Raj, Liesbet Lagae, Wim Annaert, Gustaaf Borghs
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Abstract
The present invention relates to a population of monodisperse magnetic nanoparticles with a diameter between 1 and 100 nm which are coated with a layer with hydrophilic end groups. Herein the layer with hydrophilic end groups comprises an inner layer of monosaturated and/or monounsaturated fatty acids bound to said nanoparticles and bound to said fatty acids, an outer layer of a phospholipid conjugated to a monomethoxy polyethyleneglycol (PEG) comprising a hydrophilic end group, or comprises a covalently bound hydrophilic layer bound to said nanoparticles.
Claims10
An isolated complex of a positively charged nanoparticle with a plasma membrane or an endosome, each isolated from other cellular organelles, wherein the positively charged nanoparticle comprises
- a magnetic nanoparticle coated with a layer of hydrophilic end groups, wherein
- the coated nanoparticle has a zeta potential in the range of 10-30 mV at pH 7 and a diameter between 1 and 100 nm, and
- the hydrophilic end groups comprise
- an inner layer consisting essentially of saturated and/or monounsaturated fatty acids bound to said nanoparticle and
- an outer layer of phospholipids bound to the fatty acids and conjugated to monomethoxy polyethyleneglycol (PEG),
- and wherein said nanoparticle does not comprise a peptide moiety.
A method for isolating a plasma membrane of a cell, a fraction thereof, or a plasma membrane derived organelle, comprising:
- a) providing a population of intact and suspended cells at a temperature where endocytic uptake by a cell is inhibited,
- b) contacting said intact cells with positively charged magnetic nanoparticles, thereby allowing the binding of magnetic nanoparticles to and into the cell plasma membrane, wherein the positively charged nanoparticle comprises a magnetic nanoparticle coated with a layer of hydrophilic end groups, wherein
- the coated nanoparticle has a zeta potential in the range of 10-30 mV at pH 7 and a diameter between 1 and 100 nm, and
- the hydrophilic end groups comprise
- an inner layer consisting of saturated and/or monounsaturated fatty acids bound to said nanoparticle and
- an outer layer of phospholipids bound to the fatty acids and conjugated to monomethoxy polyethyleneglycol (PEG),
- and wherein said nanoparticle does not comprise a peptide moiety,
- c) removing unbound magnetic nanoparticles,
- d) disrupting the cells,
- e) removing cellular organelles,
- f) isolating from the disrupted cells by magnetic attraction the plasma membranes with magnetic nanoparticles.
A method for isolating a plasma membrane of a cell, a fraction thereof, or a plasma membrane derived organelle, comprising:
- a) providing a population of intact and suspended cells at a temperature where endocytic uptake by a cell is inhibited,
- b) contacting said intact cells with positively charged magnetic nanoparticles, thereby allowing the binding of magnetic nanoparticles to and into the cell plasma membrane, wherein the positively charged nanoparticle comprises a magnetic nanoparticle coated with a layer of hydrophilic end groups, wherein
- the coated nanoparticle has a zeta potential in the range of 10-30 mV at pH 7 and a diameter between 1 and 100 nm, and
- the layer of hydrophilic end groups comprise hydrophilic layer covalently bound to the nanoparticles,
- and wherein said nanoparticle does not comprise a peptide moiety,
- c) removing unbound magnetic nanoparticles,
- d) disrupting the cells,
- e) removing cellular organelles,
- f) isolating from the disrupted cells by magnetic attraction the plasma membranes with magnetic nanoparticles.
An isolated complex of a positively charged nanoparticle with a plasma membrane or an endosome, each isolated from other cellular organelles, wherein the positively charged nanoparticle comprises
- a magnetic nanoparticle coated with a layer of hydrophilic end groups, wherein
- the coated nanoparticle has a zeta potential in the range of 10-30 mV at pH 7 and a diameter between 1 and 100 nm, and
- the hydrophilic end groups comprise a hydrophilic layer covalently bound to the nanoparticles,
- and wherein said nanoparticle does not comprise a peptide moiety.
Dependencies are inferred from the claim text.