The polymerisation and negative staining of Glutamic acid dehydrogenase (GDH)

The polymerisation and negative staining of Glutamic acid dehydrogenase (GDH) [1]

 

GDH, from Bovine liver, Type I, ’Sigma’, G 2501, 18 mg/ml suspension in 2.0 M (NH4)2SO4 pH 7.0

 

Reagents:

10 mM EDTA (’Sigma’, ED2SS; 372.2 g/mol): 372.2 mg/100 ml;

0.2 M NaH2PO4 + 0.2 M Na2HPO4, pH 7.0 at room temperature.

 

For the details see:

  • Josephs R., Electron microscope studies on Glutamic dehydrogenase: Subunit structure of individual molecules and linear associates, J.Mol.Biol., 1971, 55: 147-153.

  • Munn EA., Structure of oligomeric and polymeric forms of ox liver glutamate dehydrogenase examined by electron microscopy, Biochim.Biophys.Acta, 1972, 285: 301-313.

 

The protocol:

Important! Keep the buffer solution at room temperature!

Immediately prior to microscopy, dissolve the required amount of GDH in a solution of 0.2 M sodium phosphate buffer, 10 mM EDTA, pH 7.0.

The final amount of GDH in the solution has to be > 1 mg/ml. At 10 mg/ml the filaments are longer.

  1. The prep (6 l) on the grid for for 30 sec–1 min;
  2. wash with water (8 7 sec);
  3. stain with 1 uranylacetate.

 

2003 07 07 – Julius

 

The polymerisation and negative staining of Glutamic acid dehydrogenase (GDH) [2]

 

Important!

This protocol for GDH polymerisation is the same as for Catalase!

The filaments are several times longer then those formed under ’standard’ conditions for GDH polymerisation.

 

GDH, from Bovine liver, Type I, ’Sigma’, G 2501, 18 mg/ml suspension in 2.0 M (NH4)2SO4 pH 7.0

 

Reagents:

0.1 M NaH2PO4 + 0.1 M Na2HPO4, pH 6.0 at room temperature;

21.14 mg of (NH4)2SO4 (’Merck’, No. 1.01217, 132.14 g/mol).

For the details see Kiselev N.A. et al, Crystallization of Catalase in the form of tubes with monomolecular walls, J.Mol.Biol., 1967, 25: 433-441.

 

The protocol:

Dissolve the required amount of GDH in a solution of 0.1 M sodium phosphate buffer, pH 6.0, add ammonium sulphate to obtain the final concentration of 1.6 M, and leave in a fridge at +4C for 16 hr. The final amount of GDH in the solution has to be > 1 mg/ml

  1. The prep (6 l) on the grid for for 30 sec–1 min;

  2. wash with water (8 7 sec);
  3. stain with 1 uranylacetate.

 

2003 07 01 – Julius