Isolation of rafts from mouse brain tissue by a detergent-free method.
Membrane rafts are rich in cholesterol and sphingolipids and have specific proteins associated with them. Due to their small size, their identification and isolation have proved to be problematic. Their insolubility in nonionic detergents, such as Triton-X 100, at 4 degrees C has been the most common means of isolation. However, detergent presence can produce artifacts or interfere with ganglioside distribution. The direction is therefore toward the use of detergent-free protocols. We report an optimized method of raft isolation from lipid-rich brain tissue using a detergent-free method. We compared this to Triton-X 100-based isolation along sucrose or Optiprep gradients using the following endpoints: low protein content, high cholesterol content, presence of Flotillin 1 (Flot1), and absence of transferrin receptor (TfR) proteins. These criteria were met in raft fractions isolated in a detergent-free buffer along a sucrose gradient of 5%/35%/42.5%. The use of optiprep gave less consistent results with respect to protein distribution. We demonstrate that clean raft fractions with minimal myelin contamination can be reproducibly obtained in the top three low-density fractions along a sucrose step gradient.
Persaud-Sawin
Dixie-Ann
DA
Laboratory of Molecular Toxicology/Neurotoxicology Group, National Institute of Environmental Health Sciences, RTP, NC 27709, USA. sawind@niehs.nih.gov
Lightcap
Samantha
S
Harry
G Jean
GJ
D000818:Animals; D001923:Brain Chemistry; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000302:isolation & purification; D003902:Detergents; D008563:Membrane Lipids / Q000302:isolation & purification; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D009186:Myelin Sheath / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D017830:Octoxynol; D011990:Receptors, Transferrin / Q000302:isolation & purification; D013395:Sucrose
D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011990:Receptors, Transferrin; C106354:flotillins; D013395:Sucrose; D017830:Octoxynol; D002784:Cholesterol