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Pre-Absorbing Antibody with Yeast Cells Preparation of Fixed Yeast

Summary: 106
Pre-Absorbing Antibody with Yeast Cells
Preparation of Fixed Yeast
1. Plan to do steps 1-10 in the yeast immunofluorescence method. But, start with 100 mls of cells at
OD600=0.2. Then, do all steps in quadruplicate. Do pretreatment, and digest cells for 10 minutes.
2. Pool all yeast in SPC + Pics in one microfuge tube. Spin in microfuge for 30 seconds to pellet
3. Wash 3X with 1 ml of PBS + 0.1% Tween 20 (PBSTw) + 2X Pics + 1 mM EDTA. Rotate for 5
minutes at room temperature during each wash step.
4. Resuspend in 0.5 ml PBSTw + 2X Pics + 1 mM EDTA. Add 0.5 ml glycerol. Mix completely. Store
at -20C.
Preabsorption of Antibody
1. For preabsorbtion, prepare a dilution of antibody in 0.1-1.0 ml of buffer in a microfuge tube. Add
5-20 l of yeast suspension. Vortex suspension before use.
2. Mix gently for 1 hour at room temperature on end-over-end rotator. Diluted antibody plus yeast
does not need to slosh back and forth in tube. As long as yeast are suspended, preabsorption will
take place.
3. Spin in microfuge 5 minutes at top speed to remove yeast from antibody. Avoid pellet of yeast when
transferring and collecting preabsorbed antibody. If necessary, dilute antibody further to achieve
final desired concentration.


Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida


Collections: Biology and Medicine