Identification and characterization of human erythrocyte lipid raft-associated proteins.

(A) A total of 150 μL of packed red cells was extracted with 0.5% Triton X-100 on ice and centrifuged. The lipid rafts were prepared from the detergent-insoluble pellet by discontinuous density gradient centrifugation as described in method A. Twenty 150 μL fractions were collected from the top and pooled according to their contents. Aliquots of these pools were analyzed by 11% polyacrylamide gel electrophoresis/silver staining (top panel), Western blotting, and for AChE activity, as indicated. Lane 1, pellet resuspended in 300 μL TBS; lane 2, fractions 17 to 20 (high density); lane 3, fractions 9 to 16 (medium density); lane 4, fractions 7 to 8 (lipid rafts). Fractions 1 to 6 (low density, not shown) did not contain protein. (B) Lipid rafts prepared by method A were extracted with Na₂CO₃ and pelleted. Aliquots of the total sample before extraction (T), the supernatant (S), and resuspended pellet (P) were analyzed by silver staining/immunoblotting and for AChE activity. (C) Lipid rafts containing only integral proteins were prepared in one step from 100 μL of packed red cells as described in method B. The raft proteins were analyzed by gel electrophoresis/silver staining, Western blotting, and for AChE activity, as indicated. (D) Analysis of oligomeric complexes. Lipid rafts prepared by method A were solubilized in 0.5% Triton X-100 at 37°C, and 200 μL of the extract was subjected to sucrose density (5%-30%) centrifugation. Eighteen fractions were collected and analyzed by immunoblotting, as indicated. Molecular masses of marker proteins are in kilodaltons.