[32P]-labeling of Dictyostelium in phosphate-free FM
[32P]-labeling of Dictyostelium in phosphate-free FM
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Note:This protocol was developed for immunoprecipitating [32P]-labeled myosin regulatory light chain (Smith at al. 1996). It has also been successfully used for immunoprecipitation of [32P]-labeled myosin light chain kinase A, which is about 100-fold less abundant than the regulatory light chain (Smith at al. 1996).
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Procedure
- Prepare phosphate-free FM by substituting 20 mM MES, pH 6.8, for phosphate in the FM recipe.
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- Cells should be in log phase, and growing in HL5 or a 1:1 mix of HL5 and FM (prepared with phosphate). Harvest gently:
- Centrifuge at 1500 rpm for 5 min in a tabletop clinical centrifuge
- Wash once in phosphate-free FM
- Resuspend in 10 ml phosphate-free FM at 3x106 cells/ml
- Transfer to a 50 ml flask
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- Shake at 200 rpm at 22°C.
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- After 7 hr phosphate starvation, harvest 107 cells, and remove the media to leave a final volume of 0.5 ml. Resuspend the cell pellet, and transfer 200 µl aliquots to 1.5 ml screw cap tubes (Starstedt, Newton, NC).
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- Add 100 µCi of [32P]Pi (ICN, Irvine, CA) to each tube, and rotate the cultures end-over-end (8 rotations/min) for 3 hr at 22°C. We use a LabQuake "Rotisserie" (Barstead/Thermolyne, Dubuque, IA).
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- Harvest the labeled cells for 2 min in a microfuge at 2000 x g. Approximately half of the added 32P should remain with the pellet.
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