35S-methionine labeling of Dictyostelium

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. Resuspend cells in 7 ml HL5 (1x107 cells/ml) supplemented with 5 mCi of Translabel (35S-met). Shake for desired pulse time.

  3. 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).

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!


Steady state labelling

  1. To cells growing in early log phase (1x106/ml), add 50 ┬ÁCi Translabel/ml cells in HL5.

  2. Shake cells or grow on plates overnight.

  3. Pellet and wash cells with fresh, cold media or DB.

  4. Use 1 to 4x107 cells per immunoprecipitation. Keep a sample of whole cell extract to check incorporation.

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