Antibody cleanup

In order to decrease the amount of nonspecific staining, it is often necessary to preabsorb primary and secondary antibodies to yeast cells lacking the antigen prior to use. A 1:1 mixture of fixed yeast whole cells and spheroplasts are used for this purpose.

  1. Grow cells in 200 mls of YPD at 30oC to an OD 600 of 1.
  2. Add formaldehyde to the 200 ml culture to a final concentration of 3.7%. Fix for 1 hour at room temperature with shaking.
  3. Centrifuge cells at 3000 X g. Resuspend in 200 mls Solution A. Repeat wash.
  4. Pellet cells as above and resuspend in 4 mls Solution A. Reserve 2 mls of the cell suspension.
  5. Spheroplast the remaining 2 ml of the cell suspension by addition of beta-mercaptoethanol to 0.1%, glusulase to 0.05%, and zymolyase (100T) to 60 ug/ml. Incubate 1 hour at 37oC.
  6. Pellet spheroplasts as above. Resuspend in 10 mls Solution A and pellet again.
  7. Combine the suspension of whole cells with the spheroplasts and pellet as above. Resuspend in 10 mls of PBS.
  8. Put 200 ul (3 mg/ml) of the antibody to an eppendorf tube. Add 0.8 mls of PBS.
  9. Pellet 1 ml of the cell suspension and discard the PBS supernatant. Add the antibody solution to the cell pellet, gently resuspend the cells, and incubate for 1 hour on ice.
  10. Pellet cells, giving an antibody supernatant. Add this to a cell pellet prepared as in step 9. Be very careful not to get supernatants mixed up!
  11. Repeat steps 9 + 10 seven times, or as often as necessary to decrease the background.
  12. Pellet cells. Save antibody supernatant, which is now diluted 1:5 from its initial concentration. These antibodies can be stored at 4oC for several weeks or at -70oC for longer periods.

Solutions

Solution A1.2 M sorbitol, 50 mM KPO4, pH 7.0
PBS150 mM NaCl, 50 mM NaPO4, pH 7.4

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