Isolation of Dictyostelium centrosomes

Isolation of Dictyostelium centrosomes

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Improved protocol according to Gräf et al. 1998; see also Gräf 2001.

[MATERIALS] - [INDEX]

Procedure

  1. Centrifuge cell suspension in AX-medium (ca. 5 x 109 cells with a density of ca. 2 - 4 x 106 cells per ml) for 5 min at 500 x g at 4°C.

  3. Wash 3 x in phosphate buffer (ca. 5-fold volume of the pellet) spin down for 5 min at 500 x g at 4°C. Add 2 µM cytochalasin A or D prior to the last washing step.

  5. While washing, supplement 20 ml of Lysis buffer with Protease inhibitor cocktail, 2 µM cytochalasin and Triton X-100 .

  7. Resuspend cells in Lysis buffer and vortex for 1 min.

  9. Filter suspension through 5 µm Nuclepore-Filter and immediately centrifuge for 10 min at 2500 x g at 4°C.

  11. While centrifuging, complete 20 ml Pyrophosphate buffer and put it on ice.

  13. Discard supernatant and resuspend pellet in Pyrophosphate buffer, vortex for 1 min and immediately centrifuge for 10 min at 2,500 x g at 4°C.

  15. While centrifuging, prepare the following:
    • Dissolve 6 mg heparin in 600 µl water.
    • Prepare 5 µm Nuclepore-Filter.
    • Supplement 1 ml 80% sucrose and 2 ml 50% sucrose with protease inhibitor cocktail, β-mercaptoethanol and Triton X-100 .

  17. Discard pellet and add the heparin solution to the supernatant and place on ice for 5 min.

  19. Fill two Beckman SW40-tubes with 0.5 ml of supplemented 80% Sucrose solution and layer 1 ml of supplemented 50% sucrose solution above.

  21. Filtrate supernatant through Nuclepore-filter and layer one half of the filtrate on top of the density gradient in each tube.

  23. Centrifuge for 1 h at 40000 x g (21000 rpm) at 4°C.

  25. While centrifuging, prepare second density gradient:

    • Supplement Sucrose solutions with Protease inhibitor cocktail, β-mercaptoethanol and Triton X-100 : 1.05 ml 80%, 2.1 ml 55%, 2.1 ml 50% sucrose, 2 ml gradient buffer without sucrose.
    • Prepare two Beckman SW50.1 gradient tubes: each 0.5 ml 80% sucrose, 1 ml 55% sucrose and 1 ml 50% sucrose.

  27. Remove all liquid up to the upper half of the 50% fraction including the faint white band which is often visible. Resuspend all remaining liquid including the small pellet. Pass the suspension twice through a 27 gauge needle using a 1 ml syringe and supplement with the 0.5-fold volume of supplemented Gradient buffer.

  29. Load suspension onto the 2nd gradient

  31. Centrifuge for 1 h at 40,000 x g (21,000 rpm) at 4°C.

  33. Collect fractions from the bottom using a peristaltic pump and a glass capillary (ca. 200 µl per min). The glass capillary should not touch the pellet which might have formed. The volume of the first fraction which is the centrosome fraction should be ~700 µl. If there is a clear pellet it usually contains intact centrosomes as well, but these are heavily contaminated with DNA.

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Materials

  • AX-medium (1 liter)
    • 14.3 g peptone (Oxoid)
    • 7.15 g yeast extract (Oxoid)
    • 0.616 g Na2HPO4·2H2O
    • 0.486 g KH2PO4
    • 18 g glucose

    Adjust pH to 6.7 with NaOH.

  • Phosphate buffer
  • 14.6 mM KH2PO4
  • 2 mM Na2HPO4

  • Lysis buffer
  • 100 mM Na-PIPES pH 6.9
  • 2 mM MgCl2
  • 10 % sucrose

Add prior to use:

  • 0.25 % Triton X-100
  • 1 x protease inhibitor cocktail
  • 2 µM cytochalasin A

  • Pyrophosphate buffer
  • 100 mM Na-PIPES
  • 2 mM MgCl2
  • 30 % sucrose

Add prior to use:

  • 40 mM tetra-sodium diphosphate/HCl pH 6.9 (Mr=446 g/mol)
  • 1 mM DTT
  • 1 % Triton X-100
  • 1 x protease inhibitor cocktail

  • Protease inhibitor cocktail (28x; values given here refer to 1 x concentration !)
  • 1 mM Pefabloc SC 25 µg/ml leupeptin
  • 10 µg/ml tosyl-arginin-methylester
  • 10 µg/ml
  • soybean trypsin inhibitor
  • 1 µg/ml aprotinine
  • 1 µg/ml pepstatine
  • 2 mM benzamidine
  • 1 mM Na-ATP

  • Gradient buffer
  • 10 mM Na-PIPES
  • 2 mM MgCl2

Add prior to use:

  • 0.1 % Triton X-100
  • 0.1 % β-mercaptoethanol

  • Sucrose solution
  • 80% sucrose in 10 mM Na-PIPES/2 mM MgCl2

Add prior to use:

  • 0.1 % Triton X-100 0.1 % β-mercaptoethanol
  • 1x protease inhibitor cocktail

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