An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Praha 4 Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Praha 4 Czech Republic

Institute of Physiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Praha 4 Czech Republic

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Dolejškova 2155 3 182 23 Prague 8 Czech Republic

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$a The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts / $c P. Angelisová, O. Ballek, J. Sýkora, O. Benada, T. Čajka, J. Pokorná, D. Pinkas, V. Hořejší,

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$a An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

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$a Ballek, Ondřej $u Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic.

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$a Sýkora, Jan $u J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 2155/3, 182 23 Prague 8, Czech Republic.

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$a Benada, Oldřich $u Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha, 4, Czech Republic.

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$a Čajka, Tomáš $u Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic.

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$a Pokorná, Jana $u Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic.

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$a Pinkas, Dominik $u Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic.

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$a Hořejší, Václav $u Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic. Electronic address: vaclav.horejsi@img.cas.cz.

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