The polymerisation and negative staining of Actin

Actin, from Bovine muscle, ’Sigma’, A 3653,

or from Rabbit muscle, ’Molecular Probes’, A 12375 (in 0.5 ml of 2 mM Tris, pH 8.0)

 

The solutions:

10 mM Tris/HCl, pH 7.8 at room temperature: 121.1 mg/100 ml

0.2 mM DTT: 3.08 mg

1 mM MgCl2 6H2O: 20.33 mg

0.2 mM ATP-Na2 (for the stock solution of 20 mM: 11.604 mg/1 ml 10 mM Tris)

 

For the details see: Korn ED., Carlier M-F., Pantaloni D., Actin polymerization and ATP hydrolysis, Science, 1987, 238 (4827): 638-644.

or

1 mM Tris/HCl, pH 7.6 at room temperature: 12.11 mg/100 ml

100 mM KCl: 0.7456 g

1 mM MgCl2 6H2O: 20.33 mg

0.2 mM ATP-Na2 (for the stock solution of 20 mM: 11.604 mg/1 ml 1 mM Tris)

For the details see: Nagy B. and Jencks WP., Depolymerization of F-Actin by concentrated solutions of salts and denaturing agents, J.Amer.Chem.Sci., 1965, 87 (11): 2480-2488.

The final amount of actin in the solution has to be > 1 mg/ml

 

The protocol:

Mix the buffer solution ( 100 microl) and the actin, and centrifuge the solution in a microcentrifuge after 2 min (for about 10 sec at 14 000 rpm) before applying on the grid:

  1. The prep (5l) on the grid for 30 sec – 1 min

  2. wash with water

  3. stain with 1 uranylacetate.

 

2003 06 20 – Julius