³⁵S-methionine labeling of Dictyostelium

Contributed by Rex Chisholm, protocol of Cardelli lab.

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Pulse-chase

1. Harvest cells in log phase. Collect 7x10⁷ cells.
2. 
   
3. Resuspend cells in 7 ml HL5 (1x10⁷ cells/ml) supplemented with 5 mCi of Translabel (³⁵S-met). Shake for desired pulse time.
4. 
   
5. At the end of pulse period, harvest cells and resuspend in 30-50 ml unlabelled HL5. Shake and harvest 1x10⁷ cells for each chase time point (enough for 1-2 immunoprecipitations).
6. 
   

Note: there is no need to use special met-starvation media. Labeling media (5 mCi/7 ml) can be filter-sterilized and reused for up to 3 months or 8-10 labelings!

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Steady state labelling

1. To cells growing in early log phase (1x10⁶/ml), add 50 µCi Translabel/ml cells in HL5.
2. 
   
3. Shake cells or grow on plates overnight.
4. 
   
5. Pellet and wash cells with fresh, cold media or DB.
6. 
   
7. Use 1 to 4x10⁷ cells per immunoprecipitation. Keep a sample of whole cell extract to check incorporation.

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