Various compositions of lipid mixtures were prepared using the dehydration-rehydration method described by Karlsson et al. 35 A full list of the mixtures has been provided in SI 1 (ESI †). Briefly: fatty acids, phospholipids (all lipid products from Avanti Polar Lipids, USA) and phospholipid conjugated fluorophore 16 : 0 Liss Rhodamine PE (Avanti Polar Lipids, USA) were dissolved in chloroform (Merck Life Science, Norway) and methanol (VWR International) 2 : 1 mixture to a final concentration of 10 mg mL À1 . The solvent was evaporated in a rotary evaporator (Bu ¨chi, Switzerland) under reduced pressure (20 kPa) over 6 h. Subsequently, the dry lipid film was rehydrated with 3 mL of 0.2 M 8.5 pH Bicine 36 (Merck Life Science, Norway) buffer (pH adjusted with NaOH (Merck Life Science, Norway)) and 30 mL of Glycerol (Merck Life Science, Norway) to prevent complete dehydration of the lipid film and enable bilayer separation. The rehydrated lipids were kept overnight at 4 1C and thereafter swirled until there was no dry lipid residue. If swirling was not sufficient, the lipids were sonicated for 5 s.
Liss rhodamine pe
Manufactured by Avanti Polar Lipids
Sourced in United States
Liss Rhodamine PE is a lipid-soluble fluorescent dye used for labeling and tracking phospholipids in biological samples. It is a phosphatidylethanolamine (PE) conjugate of the rhodamine dye, which provides a stable and bright fluorescent signal.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Avantor (1 mentions)
Chloroform, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Rotary evaporator, by Büchi (1 mentions)
Nuclepore track etched membrane, by Cytiva (1 mentions)
Egfp antibody, by Roche (1 mentions)
E coli polar lipid, by Avanti Polar Lipids (1 mentions)
Imagequant las 4000 system, by Fujifilm (1 mentions)
Tls 55 rotor, by Beckman Coulter (1 mentions)
2 protocols using liss rhodamine pe
1
Lipid Mixture Preparation for Bilayer Studies
2
Liposome Flotation Assay for Protein Binding
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Liposome generation and flotation was performed as described in Vollmer et al., 2015 (link). In short, E. coli polar lipids (Avanti Polar Lipids) dissolved in chloroform and supplemented with 0.2 mol% 18:1 Liss Rhodamine PE (Avanti Polar Lipids) were vacuum-dried on a rotary evaporator, dissolved as liposomes in PBS by freeze/thawing cycles and extruded by passages through Nuclepore Track-Etched Membranes (Whatman) with defined pore sizes using an Avanti Mini-Extruder to generate small unilamellar liposomes of defined sizes. For liposome flotations, proteins (6 μM) were mixed 1:1 with liposomes (6 mg/mL) and floated for 2 hr at 55,000 rpm in a TLS-55 rotor (Beckman) at 25°C through a sucrose gradient. Binding efficiency was determined by Western blot analysis using an EGFP antibody (Roche, 11814460001, 1:2000). As secondary antibody, an anti-mouse, horseradish peroxidase conjugated antibody (Calbiochem, 401215, 1:5000) was used. The ImageQuant LAS-4000 system (Fuji) and the AIDA software were used to compare band intensities of start materials with floated liposome fraction.
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