I'm encountering difficulty in achieving a purified extract of erythrocyte membrane proteins using a lysis procedure with 0.1% Triton, followed by multiple centrifugation steps at 12000 g and numerous washes with the same solution. Despite these efforts, I consistently obtain a red pellet. Could someone offer assistance, please?
Simply dilute the washed erys from an outdated blood donation 10-fold into distilled, ice-cold water, let them lyse for 30 min on ice and spin down the membranes in a high-speed centrifuge (Sorvall GS3, 9000 rpm, 60 min, 4 °C). Most of the hemoglobin (and other cytosolic proteins) will be in the (deep-red) supernatant, the pellet (pure cell membranes as erys don't have organelles) should be pink. Carefully discard the supernatant (the pellet is easily disturbed) and wash the pellet twice with a dilute buffer (say, 5 mM Hepes or Tris of appropriate pH) and only then extract with detergent. If you need sealed ghosts or inside-out vesicles, see doi:10.1016/0076-6879(74)31019-1
Thank you so much Engelbert! Just one question, what do you mean by sealed ghosts or inside-out vesicles? In my case I only need to purify the membrane proteins.
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