[³²P]-labeling of Dictyostelium in phosphate-free FM

Download word file.

---

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).

---

[TOP] [INDEX]

Procedure

1. Prepare phosphate-free FM by substituting 20 mM MES, pH 6.8, for phosphate in the FM recipe.
2. 
   
3. 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
4. 
   
5. Shake at 200 rpm at 22°C.
6. 
   
7. 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).
8. 
   
9. 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).
10. 
    
11. 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.
12. 
    

[TOP] [INDEX]