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.
- Grow cells in 200 mls of YPD at 30oC to an OD 600 of 1.
- Add formaldehyde to the 200 ml culture to a final concentration of 3.7%. Fix for 1 hour at room temperature with shaking.
- Centrifuge cells at 3000 X g. Resuspend in 200 mls Solution A. Repeat wash.
- Pellet cells as above and resuspend in 4 mls Solution A. Reserve 2 mls of the cell suspension.
- 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.
- Pellet spheroplasts as above. Resuspend in 10 mls Solution A and pellet again.
- Combine the suspension of whole cells with the spheroplasts and pellet as above. Resuspend in 10 mls of PBS.
- Put 200 ul (3 mg/ml) of the antibody to an eppendorf tube. Add 0.8 mls of PBS.
- 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.
- 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!
- Repeat steps 9 + 10 seven times, or as often as necessary to decrease the background.
- 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.
|Solution A||1.2 M sorbitol, 50 mM KPO4, pH 7.0|
|PBS||150 mM NaCl, 50 mM NaPO4, pH 7.4|