Purified TmrAB and MSP1D1 were mixed with bovine brain lipid extract (Sigma-Aldrich) solubilized in 20 mM of β-DDM at a TmrAB:MSP1D1:lipid molar ratio of 1:7.5:100 in SEC buffer without detergent. After incubation at RT for 30 min, SM-2 Bio-beads™ (Bio-Rad) were added in two consecutive incubation steps to remove the detergent (1 h, overnight). TmrAB-containing nanodiscs were separated from empty discs by SEC (Superdex™ 200 Increase 3.2/300, GE Healthcare) in SEC buffer without detergent and concentrated to 5 mg/ml at 1,500 x g using Amicon® Ultra-0.5 ml centrifugal filters with a 50 kDa cut-off (Millipore). For EM, a 1.25-fold molar excess of Nb9F10 was added and incubated for 15 min on ice. TmrAB-Nb-complexes were separated from unbound Nb by SEC (KW404-4F, Shodex) in SEC buffer without detergent.
Bovine brain lipid extract
Manufactured by Merck Group
Bovine brain lipid extract is a laboratory product that contains a mixture of lipids derived from bovine brain tissue. It is a complex mixture of various phospholipids, glycolipids, and other lipid components found naturally in the brain. This product is commonly used in research applications that require a source of brain-derived lipids for experimental purposes.
Automatically generated - may contain errors
2 protocols using bovine brain lipid extract
1
Reconstitution of TmrAB Nanodiscs
2
Reconstitution of TmrAB Nanodiscs
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Purified TmrAB and MSP1D1 were mixed with bovine brain lipid extract (Sigma-Aldrich) solubilized in 20 mM of β-DDM at a TmrAB:MSP1D1:lipid molar ratio of 1:7.5:100 in SEC buffer without detergent. After incubation at RT for 30 min, SM-2 Bio-beads™ (Bio-Rad) were added in two consecutive incubation steps to remove the detergent (1 h, overnight). TmrAB-containing nanodiscs were separated from empty discs by SEC (Superdex™ 200 Increase 3.2/300, GE Healthcare) in SEC buffer without detergent and concentrated to 5 mg/ml at 1,500 x g using Amicon® Ultra-0.5 ml centrifugal filters with a 50 kDa cut-off (Millipore). For EM, a 1.25-fold molar excess of Nb9F10 was added and incubated for 15 min on ice. TmrAB-Nb-complexes were separated from unbound Nb by SEC (KW404-4F, Shodex) in SEC buffer without detergent.
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