Home

About

Advanced Search

Browse by Discipline

Scientific Societies

E-print Alerts

Add E-prints

E-print Network
FAQHELPSITE MAPCONTACT US


  Advanced Search  

 
Yeast Immunofluorescence Prepare Cells
 

Summary: Yeast Immunofluorescence
Prepare Cells:
1. Grow yeast in 25 ml YPD to OD600 = 0.2. We usually dilute a fresh overnight culture and grow
for 4-5 doublings. If cells are grown in synthetic media, skip step 2, and proceed to step 3.
2. Add 2.5 ml of 30% paraformaldehyde to 25 ml of culture while swirling. Swirl gently for 10
minutes at room temperature.
Paraformaldehyde (30%):
A. Add 2.0 g paraformaldehyde to 5 ml of dH2O in a Pyrex tube (16 X 150 mm).
B. Heat slowly with Bunsen burner to boiling. Vortex intermittently.
C. Add 0.1 ml 1M NaOH while vortexing.
D. Chill on ice. Filter (0.45 µm). Use for one day.
3. Centrifuge for 5 minutes at 2500 rpm at room temperature.
4. Resuspend pellet in 1.0 ml of 50 mM KPi, pH 6.5, 3% paraformaldehyde at room temperature.
Transfer to microfuge tube. Mix end-over-end at room temperature for 30 minutes.
5. Microfuge ~15 seconds to pellet yeast. Do step 11 now.
6. Wash 3 X 5 minutes with 1 ml 50 mM KPi, pH 6.5, 50 mM NH4Cl.
7. Pretreatment.
A. Resuspend cell pellet in 0.5 ml of freshly prepared Pretreatment Buffer at room temperature.
Pretreatment Buffer: 100 mM Tris·HCl, pH 9.0
50 mM DTT

  

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

 

Collections: Biology and Medicine